Polymorphisms in the human gene for the multidrug resistance-associated protein 1 (mrp-1) and their use in diagnostic and therapeutic applications

ABSTRACT

The present invention relates to a polymorphic MRP-1 polynucleotide, genes or vectors comprising the polynucleotides and a host cell genetically engineered with the polynucleotide or gene. Also provided are methods for producing molecular variant polypeptides, cells capable of expressing a molecular variant polypeptide and to a polypeptide encoded by the polynucleotide or the gene or obtainable by the method or cells produced herein. Also provided is an antibody to the polypeptide, a transgenic animal, and to a solid support comprising one or a plurality of the provided polynucleotides, genes, vectors, polypeptides, antibodies or host cells. Furthermore, methods of identifying a polymorphism, identifying and obtaining a pro-drug or drug or an inhibitor are also provided. In addition, the invention relates to methods for producing of a pharmaceutical composition, diagnosing a disease and, detection of the polynucleotide. Furthermore, provided herein are uses of the polynucleotides, genes, vectors, polypeptides or antibodies herein.

The present invention relates to a polymorphic MRP-1 polynucleotide.Moreover, the invention relates to genes or vectors comprising thepolynucleotides of the invention and to a host cell geneticallyengineered with the polynucleotide or gene of the invention. Further,the invention relates to methods for producing molecular variantpolypeptides or fragments thereof, methods for producing cells capableof expressing a molecular variant polypeptide and to a polypeptide orfragment thereof encoded by the polynucleotide or the gene of theinvention or which is obtainable by the method or from the cellsproduced by the method of the invention. Furthermore, the inventionrelates to an antibody which binds specifically the polypeptide of theinvention. Moreover, the invention relates to a transgenic non-humananimal. The invention also relates to a solid support comprising one ora plurality of the above mentioned polynucleotides, genes, vectors,polypeptides, antibodies or host cells. Furthermore, methods ofidentifying a polymorphism, identifying and obtaining a pro-drug or drugor an inhibitor are also encompassed by the present invention. Inaddition, the invention relates to methods for producing of apharmaceutical composition and to methods of diagnosing a disease.Further, the invention relates to a method of detection of thepolynucleotide of the invention. Furthermore, comprised by the presentinvention are a diagnostic and a pharmaceutical composition. Even more,the invention relates to uses of the polynucleotides, genes, vectors,polypeptides or antibodies of the invention. Finally, the inventionrelates to a diagnostic kit.

The human multidrug resistance-associated protein (MRP) family, asubfamily of the ATP-binding cassette (ABC) protein superfamily,currently contains seven members. ABC proteins are composed oftransmembrane domains (TMD's), and nucleotide binding domains (NBD's, orATP-binding cassettes). The ability of several of these membraneproteins to transport a wide range of anticancer drugs out of cells andtheir expression in many tumor types, make them to possible candidatesinvolved in unexplained cases of drug resistance (Borst et al. 2000, JNatl Cancer Inst 92 (16): 1295-1302; Borst et al. 1999, Biochimica etBiophysica Acta 1461: 347-357; Klein et al. 1999, Biochimica etBiophysica Acta 1461: 237-262).

One member of the human MRP family is MRP-1. The gene spans at least 200kb and contains 31 exons. Several alternatively spliced variants of theMRP-1 mRNA could be characterized. The MRP-1 gene, encodes an integralmembrane protein of 190 kDa whose function is the energy dependentexport of substances from the inside of cells, and from membranes, tothe outside. In contrast to P-glycoprotein that is invariably located inthe apical membrane of epithelial cells, MRP-1 is located basolaterallyand, therefore, tends to pump drugs into the body. The protein ispresent in many normal tissues and occurs mainly in lung, testis andmuscle and very low in liver. The MRP-1 protein is located in plasmamembranes in different tissues, like kidney and liver (Grant et al.1997, Genomics 45: 368-378; Klein et al. 1999, Biochimica et BiophysicaActa 1461, 237-262; Cole and Deeley 1998, BioEssays 20: 931-940; Borstet al. 1999, Biochimica et Biophysica Acta 1461: 347-357). In additionit could be shown, that beside P-glycoprotein likewise MRP-1 isexpressed in the epithelia of the choroid plexus (CP), in which theblood-cerebrospinal-fluid (CSF) drug permeability barrier is localized.The function of this blood-brain barrier is to isolate the brain fromcirculating drugs, toxins and xenobiotics. MRP-1 contributes to thebasolateral broad-specificity drug-permeation barrier in CP (Rao et al.1999, Proc. Natl. Acad. Sci. USA 96: 3900-3905).

In contrast to P-glycoprotein and to other members of the MRP family(MRP-4 and MRP-5), e.g. MRP-2 and MRP-1 possesses an additionalN-terminal transmembrane domain (TMD0). Thus, these proteins contain twocharacteristic hydrophilic, cytosolic ATP-binding domains (NBD's) and 3hydrophobic transmembrane domains, which include totally 17transmembrane segments. This is designated as TMD0(TMD-ABC)2 arrangement(Klein et al. 1999, Biochimica et Biophysica Acta 1461: 237-262). TheNBD's are characterized by two sequence motifs, designated “Walker A”and “Walker B”. Mutational analysis of a number of ABC proteinsindicates that these two regions are critical for ATPase function(Walker et al. 1982, EMBO J. 1: 945-951; Schneider et al. 1998, FEMSMicrobiol. Rev. 22: 1-20). Within the Walker A motif there exists aconserved lysine residue (GX₄GKS/T), which is essential in bothnucleotide binding domains for full transport function. This isconsistent with the role of this consensus sequence as the amino acidacceptor site of the phosphoryl moiety of the nucleotide. In addition,ABC transporters possess a characteristic conserved “active transportfamily” signature (or “C”) motif encompassing 14 amino acids(LSSGGQX₃RHydXHydA). This region is located between the Walker A and Bmotifs. A possible significance of this motif referring to the bindingand hydrolysis of nucleotide could be deduced from the observation, thatit is highly conserved in NBD1, but not in NBD2 of the MRP-relatedproteins. This is in contrast to observations, which point to ainvariant nature of this motif in NBD1 and NBD2 in P-glycoproteins (Coleand Deeley 1998, BioEssays 20: 931-940).

MRP-1 and the other members of the MRP family all contain a highlyconserved “deletion” of 13 amino acids located between the Walker A andB motifs in NBD1, which alters the spacing between the two Walker motifsin the first nucleotide binding domain. Recent studies have shown, thatthis deletion affects the folding and activity of this domain (Hipfneret al. 1999, J. Biol. Chem. 274 (22): 15420-6). In contrast to theNBD's, the transmembrane domains of the ABC transporters are highlydivergent. This sequence divergence is consistent with the notion thatthe transmembrane domains are important determinants of the differentsubstrate specificities of various ABC transporters (Ueda et al. 1997,Semin. Cancer Biol. 8 (3): 151-159; Hrycyna et al. 1998, J. Biol. Chem.273 (27): 16631-4). The study of post-translational modification of theMRP-1 protein by limited proteolysis and site-directed mutagenesisrevealed, that the protein is glycosylated at Asn 19 and Asn 23 in theNH2-terminal transmembrane domain and at Asn 1006 in the COOH-proximaltransmembrane domain (Hipfner et al. 1997, J. Biol. Chem. 272 (38):23623-30). Interestingly, recent studies of deletion mutants of MRP-1,by the removal of the full TMD0 region, indicated that this region isneither required for the transport function of MRP-1 nor for its properrouting to the lateral plasma membrane compartment (Bakos et al. 1998,J. Biol. Chem. 273: 32167-32175).

The members of the MRP family transport anionic drugs, likemethotrexate, neutral drugs conjugated to acidic ligands, such asglutathione (GSH), glucuronate, or sulfate. While for MRP-2 the majorphysiologic function is the transport of bilirubin glucuronides andother organic anions from liver into bile, for MRP-1 it is the transportof the cysteinyl leukotriene LTC₄. This is an important chemicalmediator of inflammatory responses in receptor-mediated signaltransduction pathways that control vascular permeability and smoothmuscle contraction. So far no major physiologic function is known forthe other members of the MRP family. MRP-1, -2 and -3 can additionallycause resistance to neutral organic drugs that are not known to beconjugated to acidic ligands by transporting these drugs together withfree GSH (Borst et al. 2000, J Natl Cancer Inst 92 (16): 1295-1302;Hipfner et al. 1999, Biochimica et Biophysica Acta 1461: 359-376).Although MRP-1, MRP-2 and MRP-3 have many common substrates, the threetransport proteins may differ in their relative affinities forindividual compounds. LTC₄ remains the highest affinity substrate knownfor MRP-1. In addition to the cysteinyl leukotriene LTC₄ many of theidentified endogenous MRP-1 substrates, like glutathione disulfide(GSSG) or bilirubin glucuronides are well characterized MRP-2 substrates(Heijn et al. 1997, Biochim. Biophys. Acta 1326: 12-22; Jedlitschky etal. 1997, Biochem. J. 327: 305-310). Beside LTC₄ the preferredsubstrates of MRP-1 are organic anions, like drugs conjugated toglutathione (GSH), glucuronate, or sulfate. MRP-1 transports for examplesubstrates, such as methotrexate (MTX) or arsenite (H₃AsO₃). Likewise avariety of other GSH-conjugated xenobiotics, including conjugates of theactivated forms of the potent carcinogen aflatoxin B1 can be activelytransported by MRP-1, suggesting a protective role of MRP-1 in chemicalcarcinogenesis (Loe et al. 1997, Mol. Pharmacol. 51 (6): 1034-41). Incontrast to that, P-glycoprotein has a low affinity for such negativelycharged compounds.

Glutathione conjugation by GSTs and transport of glutathioneS-conjugates out of cells into the extracellular space by MRP-1 havebeen shown to work as a system in the detoxification of many xenobioticsamong them many anticancer drugs. (Zhang et al., 1998, Int J Onc 12:871-882). Because of that, the degree of expression and thefunctionality of the MRP-1 gene product can affect the therapeuticeffectiveness of such agents. This is of particular importance in cancertherapy where high MRP-1, as well as P-gp expression and activitycorrelate with the resistance of cancer cells against chemotherapeuticdrugs (Gottesman et al. 1996, Curr. Biol. 6: 610-617; Nooter and Stoter1996, Path. Res. Pract. 192: 768-780).

Utilization of chemotherapy for the treatment of tumors can be limitedby its hematological toxicity. Transduction of hematopoietic progenitorswith the multidrug resistance 1 (MDR-1) or with the MRP-1 gene shouldprovide protection from toxic effects of chemotherapeutic agents. Theinterest in the use of MRP-1 as an alternative to MDR-1 for bone marrowprotection lies in its different modulation. Because MRP-1 expression isnot reversed by agents, that decrease MDR-1 tumor resistance, thesereversal agents can be used without reversing bone marrow (BM)protection of the MRP-1 transduced hematopoietic cells. These transducedcells have shown increased resistance to doxorubicin, vincristine andetoposide. In mice, a retrovirus-mediated MRP-1 gene transfer intohematopoietic cells leads to a protection from chemotherapy-inducedleukopenia (Machiels et al. 1999, Hum Gene Ther 10 (5): 801-11; D'Hondtet al. 1997, Hum Gene Ther 8 (15): 1745-51).

For understanding the physiological mechanisms of action of MRP-1, suchas mechanisms by which MRP-1 transports compounds and mediates multidrugresistance, mrp-1 knockout models in vitro, as well as in vivo have beengenerated (Wijnholds et al. 1997, Nat Med 3: 1275-1279). Because boththe human and murine MRP-1 have an 88% amino acid identity and both caninduce multidrug resistance when their respective cDNA's are transfectedinto drug-sensitive cells, it is conceivable that results from knockoutstudies can be transferred to humans (Stride et al. 1997, Mol Pharmacol52: 344-353). A total block of the murine mrp-1 has been found to becompatible with life, suggesting that MRP-1 inhibitors can be safelyused for treating cancer patients. The studies with mrp-1 knockout micehave given detailed insights in the MRP-1 transport characteristics, sothat this protein catalyzes both the export of certainglutathione-S-conjugates and a cotransport of GSH and drugs orendogenous metabolites (Rappa et al. 1999, Biochem Pharmacol 58:557-562).

Different forms of multidrug resistance (MDR) have been characterized.The classical MDR is defined by overexpression of P-glycoprotein, whilethe non-Pgp MDR phenotype has typically no expression of P-glycoprotein,but is caused by an overexpression of MRP-1. Such an overexpression hasbeen observed so far in multidrug-resistant cell lines derived from manydifferent tissue and tumor types, including both small cell and largecell lung cancer, carcinomas of the colon, breast, bladder, prostate,thyroid and cervix, glioma, neuroblastoma, fibrosarcoma, and variousforms of leukemia (Hipfner et al. 1999, Biochimica et Biophysica Acta1461: 359-376). Furthermore a cell line from renal cell carcinoma (RCC)could be established, which show resistance to adriamycin andepirubicin, in addition the cells demonstrated cross-resistance tocisplatin and 5-fluororubicin. Beside elevated MDR-1, GST-pi andtopoisomerase II mRNA levels, likewise the mRNA content for MRP-1 washigher than in a control cell line (Yu et al. 2000, Urol. Res. 28 (2):86-92).

Multidrug resistance caused by MRP-1 and P-gp is characterized by anATP-dependent reduction in drug accumulation. In respect to the drugresistance profiles of transfected cells, which overexpress P-gp orMRP-1 it could be shown that the substrate specificity of MRP-1 andP-glycoprotein is similar.

MRP-1 transfected mammalian cells are resistant to anthracyclines, suchas doxorubicin and daunorubicin, to vinca alkaloids, such as vincristineand to the etoposide VP-16. The transfected cells accumulate lowerlevels of these drugs than do control cells (Zhu et al. 1997, Oncol.Res. 9: 229-236). In addition resistance to the vinca alkaloidvinblastine, to colchicine and to the taxane paclitaxel have beenobserved, but to a rather lower extent in MRP-1 transfected cells thanin P-gp overexpressing cells. The basis of this differential sensitivityis still unknown. MRP-1 also confers resistance to certain antimonialand arsenical oxyanions (Cole et al. 1994, Cancer Res. 54: 5902-10).

Considerable interest exists in elucidating the potential involvement ofMRP-1 in clinical MDR. For the analysis of the MRP-1 expression levelsand its localization within both normal and malignant tissues, a numberof different MRP-1 antibodies have been used in immunoassays (Flens etal. 1994, Cancer Res. 54 (17): 4557-63; Hipfner et al. 1994, Cancer Res.54 (22): 5788-92). The expression of the MRP1 protein and/or mRNA hasbeen detected in almost every tumor type examined. In the following someexamples of the tumor types, which were analyzed: solid tumors, such aslung tumors, neuroblastoma, melanoma, retinoblastoma, breast andprostate cancer, as well as hematological malignancies (Takebayashi etal. 1998, Cancer 82 (4): 661-666; Campling et al. 1997, Clin. CancerRes. 3 (1): 115-22; Sullivan et al. 1998, Clin. Cancer Res. 4 (6):1393-1403; Filipits et al. 1997, Leukemia 11 (7): 1073-7). Among thecommon tumor types, expression of high levels of MRP1 is particularlyfrequent in the major histologic forms of non-small cell lung cancer.These studies suggest that MRP1 may be involved in multidrug resistanceof some tumor types or subgroups of patients, but up to now nocomprehensive picture of the general revelance of this protein toclinical multidrug resistance has defined (Hipfner et al. 1999,Biochimica et Biophysica Acta 1461: 359-376).

Nevertheless several studies have detected MRP-1 expression levels to beof prognostic significance. In childhood neuroblastoma it could beshown, that the amplification of the N-myc oncogene is a powerfulindicator of poor response to chemotherapy and poor outcome. Theanalysis of neuroblastoma tumor samples revealed significantly higherMRP-1 mRNA levels in tumors with N-myc amplification, than in tumorswithout such an amplification. In addition a correlation between levelsof MRP-1 mRNA and a reduced survival rate independent of the N-mycamplification could be found (Norris et al. 1996, N. Engl. J. Med. 334(4): 231-8).

The potential role of drug transporters in clinical multidrug resistancehas lead to a search for strategies, which allow either an inhibition ofthese drug pumps, or a reduction of the expression in cancer patients.In respect to MRP's the attempts to find inhibitors have concentrated toMRP-1 and MRP-2. Examples of potent competitive inhibitors are highaffinity substrates, such as leukotriene C4, S-decylglutathione and theleukotriene D4 anatgonist MK571. Other inhibitors are organic acids,such as probenecid and benzobromarone, which were originally developedto inhibit transport of uric acid (Borst et al. 2000, J Natl Cancer Inst92 (16): 1295-1302). Furthermore experiments using polarized cell linesand ovarian carcinoma cells, both stably expressing MRP-1 cDNA haverevealed, that V-104 (a pipecolinate derivative) partially inhibitsdaunorubicin transport by MRP-1. In addition this agent reversesetoposide resistance of MRP-1 expressing ovarian cancer cells (Evers etal. 2000, Br. J. Cancer 83 (3): 366-74). Another promising strategy forovercoming MRP-1 induced multidrug resistance is to use antisenseoligonucleotides against this drug transporter. In MRP-1 transfectedHeLa cells the treatment with an antisense oligonucleotide, targeted tothe coding region region of the MRP-1 mRNA results in a greater than 90%reduction of the MRP-1 mRNA level. Under these conditions an increasedsensitivity to doxorubicin was observed (Stewart et al. 1996, Biochem.Pharmacol. 51 (4): 461-9). The findings concerning these two strategieshave potential implications for the treatment of drug-resistant tumors.

Thus, means and methods for diagnosing and treating a variety ofdiseases and disorders based on dysfunctions or dysregulations of drugtransport were not available yet but are nevertheless highly desirable.Thus, the technical problem underlying the present invention is tocomply with the above specified needs.

The solution to this technical problem is achieved by providing theembodiments characterized in the claims.

Accordingly, the present invention relates to a polynucleotidecomprising a polynucleotide selected from the group consisting of:

-   -   (a) a polynucleotide having the nucleic acid sequence of SEQ ID        NO: 75, 76, 81, 82, 87, 88, 93, 94, 99, 100, 105, 106, 111, 112,        117, 118, 123, 124, 129, 130, 135, 136, 141, 142, 147, 148, 153,        154, 159, 160, 165, 166, 171, 172, 177, 178, 183, 184, 189, 190,        195, 196, 201, 202, 207, 208, 213, 214, 219, 220, 225, 226, 231,        232, 237, 238, 243, 244, 249, 250, 255, 256, 261, 262, 267, 268,        273, 274, 279, 280, 285, 286, 291, 292, 297, 298, 303, 304, 309,        310, 315, 316, 321, 322, 329, 330, 333, 334, 337, 338, 341, 342,        345, 346, 349, 350, 353, 354, 357, 358, 361, 362, 365, 366, 369,        370, 373, 374, 377, 378, 381, 382, 385, 386, 389, 390, 393, 394,        397 or 398;    -   (b) a polynucleotide encoding a polypeptide having the amino        acid sequence of SEQ ID NO: 324, 326, 328, 401 or 403;    -   (c) a polynucleotide capable of hybridizing to a MRP-1 gene,        wherein said polynucleotide is having a substitution or deletion        of at least one nucleotide at a position corresponding to        position 124667 of the MRP-1 gene (Accession No: AC026452),        1884, 1720 to 1723, 1163, 926, 437, 381, 233, 189, 440 or 1625        of the MRP-1 gene (Accession No: U07050), 39508 of the MRP-1        gene (GI No: 7209451), 79, 88 or 249 of the MRP-1 gene        (Accession No: AF022830), 95 or 259 of the MRP-1 gene (Accession        No: AF022831), 57998, 57853 or 53282 of the MRP-1 gene (GI No:        7209451), 137710, 137667, 38646 or 137647 of the MRP-1 gene        (Accession No: AC026452), 27159, 27258, 34206 to 34207, 34218,        34215, 55156 or 55472 of the MRP-1 gene (Accession No:        AC003026), 14008, 17970, 18195, 21133, 18067, 17900 of the MRP-1        gene (Accession No: U91318), or 150727 or 33551 of the MRP-1        gene (Accession No: AC025277), 174 of the MRP-1 gene (Accession        No: AF022828), 248 or 258 of the MRP-1 gene (Accession No:        AF022829), 51798 or 50892 of the MRP-1 gene (Accession No: GI        3582311), 37971 of the MRP-1 gene (Accession No: GI 7363401),        55296, 55132, 55114, 55112 or 20097 to 20099 of the MRP-1 gene        (Accession No: GI 2815549), 109 to 122, 76 to 78, 73 to 78, 70        to 78, 67 to 78 or 58 to 78 of the MRP-1 gene (Accession No: GI        4826837), 60357, 61786 or 39541 of the MRP-1 gene (Accession No:        GI 7209451) or a insertion of at least one nucleotide at a        position corresponding to position 55156/55157 of the MRP-1 gene        (Accession No: AC003026), 437/438 or 926/927 of the MRP-1 gene        (Accession No: U07050) or 76437/76438 of the MRP-1 gene        (Accession No: GI 7209451);    -   (d) a polynucleotide capable of hybridizing to a MRP-1 gene,        wherein said polynucleotide is having at a position        corresponding to position 124667 of the MRP-1 gene (Accession        No: AC026452) a C, at a position corresponding to position 1884        of the MRP-1 gene (Accession No: U07050) a A, at a position        corresponding to position 1720 to 1723 of the MRP-1 gene        (Accession No: U07050) a deletion, at a position corresponding        to position 1163 of the MRP-1 gene (Accession No: U07050) a T,        at a position corresponding to position 926/927 of the MRP-1        gene (Accession No: U07050) a insertion, at a position        corresponding to position 437/438 of the MRP-1 gene (Accession        No: U07050) a insertion, at a position corresponding to position        381 of the MRP-1 gene (Accession No: U07050) a G, at a position        corresponding to position 233 of the MRP-1 gene (Accession No:        U07050) an A, at a position corresponding to position 189 of the        MRP-1 gene (Accession No: U07050) an A, at a position        corresponding to position 39508 of the MRP-1 gene (GI        No: 7209451) an A, at a position corresponding to position 174        of the MRP-1 gene (Accession No: AF022828) a T, at a position        corresponding to position 248 of the MRP-1 gene (Accession No:        AF022829) an A, at a position corresponding to position 258 of        the MRP-1 gene (Accession No: AF022829) a G, at a position        corresponding to position 79 of the MRP-1 gene (Accession No:        AF022830) an A, at a position corresponding to position 88 of        the MRP-1 gene (Accession No: AF022830) a C, at a position        corresponding to position 249 of the MRP-1 gene (Accession No:        AF022830) a G, at a position corresponding to position 95 of the        MRP-1 gene (Accession No: AF022831) a C, at a position        corresponding to position 259 of the MRP-1 gene (Accession No:        AF022831) a G, at a position corresponding to position 57998 of        the MRP-1 gene (GI No: 7209451) a T, at a position corresponding        to position 57853 of the MRP-1 gene (GI No: 7209451) a T, at a        position corresponding to position 53282 of the MRP-1 gene (GI        No: 7209451) a G, at a position corresponding to position 137710        of the MRP-1 gene (Accession No: AC026452) a G, at a position        corresponding to position 137667 of the MRP-1 gene (Accession        No: AC026452) a T, at a position corresponding to position        137647 of the MRP-1 gene (Accession No: AC026452) a T, at a        position corresponding to position 27159 of the MRP-1 gene        (Accession No: AC003026) a C, at a position corresponding to        position 27258 of the MRP-1 gene (Accession No: AC003026) an A,        at a position corresponding to position 34206 to 34207 of the        MRP-1 gene (Accession No: AC003026) a deletion, at a position        corresponding to position 34215 of the MRP-1 gene (Accession No:        AC003026) a C, at a position corresponding to position        55156/55157 of the MRP-1 gene (Accession No: AC003026) a        insertion, at a position corresponding to position 55472 of the        MRP-1 gene (Accession No: AC003026) a C, at a position        corresponding to position 14008 of the MRP-1 gene (Accession No:        U91318) an A, at a position corresponding to position 150727 of        the MRP-1 gene (Accession No: AC025277) an A, at a position        corresponding to position 17970 of the MRP-1 gene (Accession No:        U91318) a deletion, at a position corresponding to position        18195 of the MRP-1 gene (Accession No: U91318) an A, at a        position corresponding to position 21133 of the MRP-1 gene        (Accession No: U91318) an A, at a position corresponding to        position 34218 of the MRP-1 gene (Accession No: AC003026) an A,        at a position corresponding to position 18067 of the MRP-1 gene        (Accession No: U91318) a T, at a position corresponding to        position 440 of the MRP-1 gene (Accession No: U07050) a T, at a        position corresponding to position 1625 of the MRP-1 gene        (Accession No: U07050) an A, at a position corresponding to        position 17900 of the MRP-1 gene (Accession No: U91318) a T, at        a position corresponding to position 38646 of the MRP-1 gene        (Accession No: AC026452) a C, at a position corresponding to        position 33551 of the MRP-1 gene (Accession No: AC025277) an A,        at a position corresponding to position 51798 of the MRP-1 gene        (Accession No: 3582311) an G, at a position corresponding to        position 37971 of the MRP-1 gene (Accession No: 7363401) an A,        at a position corresponding to position 50892 of the MRP-1 gene        (Accession No: 3582311) an A, at a position corresponding to        position 55296 of the MRP-1 gene (Accession No: 2815549) an A,        at a position corresponding to position 55132 of the MRP-1 gene        (Accession No: 2815549) an A, at a position corresponding to        position 55114 of the MRP-1 gene (Accession No: 2815549) an G,        at a position corresponding to position 55112 of the MRP-1 gene        (Accession No: 2815549) an G, at a position corresponding to        position 109 to 122 of the MRP-1 gene (Accession No: 4826837)        deletions, at a position corresponding to position 76 to 78 of        the MRP-1 gene (Accession No: 4826837) deletions, at a position        corresponding to position 73 to 78 of the MRP-1 gene (Accession        No: 4826837) deletions, at a position corresponding to position        70 to 78 of the MRP-1 gene (Accession No: 4826837) deletions, at        a position corresponding to position 67 to 78 of the MRP-1 gene        (Accession No: 4826837) deletions, at a position corresponding        to position 58 to 78 of the MRP-1 gene (Accession No: 4826837)        deletions, at a position corresponding to position 20097 to        20099 of the MRP-1 gene (Accession No: 2815549) deletions, at a        position corresponding to position 60357 of the MRP-1 gene        (Accession No: 7209451) a T, at a position corresponding to        position 61786 of the MRP-1 gene (Accession No: 7209451) an A,        at a position corresponding to position 76437/76438 of the MRP-1        gene (Accession No: 7209451) an insertion or at a position        corresponding to position 39541 of the MRP-1 gene (Accession        No: 7209451) an A;    -   (e) a polynucleotide encoding an MRP-1 polypeptide or fragment        thereof, wherein said polypeptide comprises an amino acid        substitution at position 329, 433 or 723 of the MRP-1        polypeptide (Accession No: P33527) or 73 or 989 of the MRP-1        polypeptide (Accession No: GI 2828206); and    -   (f) a polynucleotide encoding an MRP-1 polypeptide or fragment        thereof, wherein said polypeptide comprises an amino acid        substitution of Phe to Cys at position 329, Arg to Ser at        position 433 or Arg to Gln at position 723 of the MRP-1        polypeptide (Accession No: P33527) or Thr to Ile at position 73        or Ala to Thr at position 989 of the MRP-1 polypeptide        (Accession No: GI 2828206).

In the context of the present invention the term “polynucleotides” orthe term “polypeptides” refers to different variants of a polynucleotideor polypeptide. Said variants comprise a reference or wild type sequenceof the polynucleotides or polypeptides of the invention as well asvariants which differ therefrom in structure or composition. Referenceor wild type sequences for the polynucleotides are Accession No: U07050,AF022828, AF022829, AF022830, AF022831, AC026452, AC003026, U91318,AC025277 or GI No: 7209451. Reference or wild type sequence for thepolypeptides of the invention is Accession No: P33527. The differencesin structure or composition usually occur by way of nucleotide or aminoacid substitution(s), addition(s) and/or deletion(s). Preferreddeletions in accordance with the invention are a GGTA deletion at aposition corresponding to position 1720 to 1723 of the MRP-1 gene(Accession No: U07050), an AT deletion at a position corresponding toposition 34206 to 34207 of the MRP-1 gene (Accession No: AC003026) or aT deletion at a position corresponding to position 17970 of the MRP-1gene (Accession No: U91318), preferred insertions are a TCCTTCCinsertion at a position corresponding to position 437/438 of the MRP-1gene (Accession No: U07050), a TGGGGC insertion at a positioncorresponding to position 55156/55157 of the MRP-1 gene (Accession No:AC003026) or a T insertion at a position corresponding to position926/927 of the MRP-1 gene (Accession No: U07050). Preferably, saidnucleotide substitution(s), addition(s) or deletion(s) comprised by thepresent invention result(s) in one or more changes of the correspondingamino acid(s) of the polypeptides of the invention. The variantpolynucleotides and polypeptides also comprise fragments of saidpolynucleotides or polypeptides of the invention. The polynucleotidesand polypeptides as well as the aforementioned fragments thereof ofthe'present invention are characterized as being associated with a MRP-1dysfunction or dysregulation comprising, e.g., insufficient and/oraltered drug uptake. Said dysfunctions or dysregulations referred to inthe present invention cause a disease or disorder or a prevalence forsaid disease or disorder. Preferably, as will be discussed below indetail, said disease is cancer or diseases related to multidrugresistance or any other disease caused by a dysfunction or dysregulationdue to a polynucleotide or polypeptides of the invention, also referredto as MRP-1 gene associated diseases in the following.

The term “hybridizing” as used herein refers to polynucleotides whichare capable of hybridizing to the polynucleotides of the invention orparts thereof which are associated with a MRP-1 dysfunction ordysregulation. Thus, said hybridizing polynucleotides are alsoassociated with said dysfunctions and dysregulations. Preferably, saidpolynucleotides capable of hybridizing to the polynucleotides of theinvention or parts thereof which are associated with MRP-1 dysfunctionsor dysregulations are at least 70%, at least 80%, at least 95% or atleast 100% identical to the polynucleotides of the invention or partsthereof which are associated with MRP-1 dysfunctions or dysregulations.Therefore, said polynucleotides may be useful as probes in Northern orSouthern Blot analysis of RNA or DNA preparations, respectively, or canbe used as oligonucleotide primers in PCR analysis dependent on theirrespective size. Also comprised by the invention are hybridizingpolynucleotides which are useful for analysing DNA-Protein interactionsvia, e.g., electrophoretic mobility shift analysis (EMSA). Preferably,said hybridizing polynucleotides comprise at least 10, more preferablyat least 15 nucleotides in length while a hybridizing polynucleotide ofthe present invention to be used as a probe preferably comprises atleast 100, more preferably at least 200, or most preferably at least 500nucleotides in length.

It is well known in the art how to perform hybridization experimentswith nucleic acid molecules, i.e. the person skilled in the art knowswhat hybridization conditions s/he has to use in accordance with thepresent invention. Such hybridization conditions are referred to instandard text books such as Molecular Cloning A Laboratory Manual, ColdSpring Harbor Laboratory (1989) N.Y. Preferred in accordance with thepresent inventions are polynucleotides which are capable of hybridizingto the polynucleotides of the invention or parts thereof which areassociated with a MRP-1 dysfunction or dysregulation under stringenthybridization conditions, i.e. which do not cross hybridize to unrelatedpolynucleotides such as polynucleotides encoding a polypeptide differentfrom the MRP-1 polypeptides of the invention.

The term “corresponding” as used herein means that a position is notonly determined by the number of the preceding nucleotides and aminoacids, respectively. The position of a given nucleotide or amino acid inaccordance with the present invention which may be deleted, substitutedor comprise one or more additional nucleotide(s) may vary due todeletions or additional nucleotides or amino acids elsewhere in the geneor the polypeptide. Thus, under a “corresponding position” in accordancewith the present invention it is to be understood that nucleotides oramino acids may differ in the indicated number but may still havesimilar neighboring nucleotides or amino acids. Said nucleotides oramino acids which may be exchanged, deleted or comprise additionalnucleotides or amino acids are also comprised by the term “correspondingposition”. Said nucleotides or amino acids may for instance togetherwith their neighbors form sequences which may be involved in theregulation of gene expression, stability of the corresponding RNA or RNAediting, as well as encode functional domains or motifs of the proteinof the invention.

By, e.g., “position 1720 to 1723” it is meant that said polynucleotidecomprises one or more deleted nucleotides which are deleted betweenpositions 1720 and position 1723 of the corresponding wild type versionof said polynucleotide. The same applies mutatis mutandis to all otherposition numbers referred to in the above embodiment which are draftedin the same format.

By, e.g., “position 437/438” it is meant that said polynucleotidecomprises one or more additional nucleotide(s) which are insertedbetween positions 437 and position 438 of the corresponding wild typeversion of said polynucleotide. The same applies mutatis mutandis to allother position numbers referred to in the above embodiment which aredrafted in the same format, i.e. two consecutive position numbersseparated by a slash (/).

In accordance with the present invention, the mode and populationdistribution of genetic variations in the MRP-1 gene has been analyzedby sequence analysis of relevant regions of the human said gene frommany different individuals. It is a well known fact that genomic DNA ofindividuals, which harbor the individual genetic makeup of all genes,including the MRP-1 gene, can easily be purified from individual bloodsamples. These individual DNA samples are then used for the analysis ofthe sequence composition of the alleles of the MRP-1 gene that arepresent in the individual which provided the blood sample. The sequenceanalysis was carried out by PCR amplification of relevant regions ofsaid genes, subsequent purification of the PCR products, followed byautomated DNA sequencing with established methods (e.g. ABIdyeterminator cycle sequencing).

One important parameter that had to be considered in the attempt todetermine the individual genotypes and identify novel variants of theMRP-1 gene by direct DNA-sequencing of PCR-products from human bloodgenomic DNA is the fact that each human harbors (usually, with very fewabnormal exceptions) two gene copies of each autosomal gene (diploidy).Because of that, great care had to be taken in the evaluation of thesequences to be able to identify unambiguously not only homozygoussequence variations but also heterozygous variations. The details of thedifferent steps in the identification and characterization of novelpolymorphisms in the MRP-1 gene (homozygous and heterozygous) aredescribed in the Examples below.

Over the past 20 years, genetic heterogeneity has been increasinglyrecognized as a significant source of variation in drug response. Manyscientific communications (Meyer, Ann. Rev. Pharmacol. Toxicol. 37(1997), 269-296 and West, J. Clin. Pharmacol. 37 (1997), 635-648) haveclearly shown that some drugs work better or may even be highly toxic insome patients than in others and that these variations in patient'sresponses to drugs can be related to molecular basis. This“pharmacogenomic” concept spots correlations between responses to drugsand genetic profiles of patient's (Marshall, Nature Biotechnology, 15(1997), 954-957; Marshall, Nature Biotechnology, 15 (1997), 1249-1252).In this context of population variability with regard to drug therapy,pharmacogenomics has been proposed as a tool useful in theidentification and selection of patients which can respond to aparticular drug without side effects. This identification/selection canbe based upon molecular diagnosis of genetic polymorphisms by genotypingDNA from leukocytes in the blood of patient, for example, andcharacterization of disease (Bertz, Clin. Pharmacokinet. 32 (1997),210-256; Engel, J. Chromatogra. B. Biomed. Appl. 678 (1996), 93-103).For the founders of health care, such as health maintenanceorganizations in the US and government public health services in manyEuropean countries, this pharmacogenomics approach can represent a wayof both improving health care and reducing overheads because there is alarge cost to unnecessary drugs, ineffective drugs and drugs with sideeffects.

The mutations in the variant genes of the invention sometime result inamino acid deletion(s), insertion(s) and in particular insubstitution(s) either alone or in combination. It is of course alsopossible to genetically engineer such mutations in wild type genes orother mutant forms. Methods for introducing such modifications in theDNA sequence of said genes are well known to the person skilled in theart; see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, ColdSpring Harbor Laboratory (1989) N.Y.

For the investigation of the nature of the alterations in the amino acidsequence of the polypeptides of the invention may be used such asBRASMOL that are obtainable from the Internet. Furthermore, foldingsimulations and computer redesign of structural motifs can be performedusing other appropriate computer programs (Olszewski, Proteins 25(1996), 286-299; Hoffman, Comput. Appl. Biosci. 11 (1995), 675-679).Computers can be used for the conformational and energetic analysis ofdetailed protein models (Monge, J. Mol. Biol. 247 (1995), 995-1012;Renouf, Adv. Exp. Med. Biol. 376 (1995), 37-45). These analysis can beused for the identification of the influence of a particular mutation onbinding and/or transport of drugs.

Usually, said amino acid deletion, addition or substitution in the aminoacid sequence of the protein encoded by the polynucleotide of theinvention is due to one or more nucleotide substitution, insertion ordeletion, or any combinations thereof. Preferably said nucleotidesubstitution, insertion or deletion may result in an amino acidsubstitution of F to C at position corresponding to position 329 of theMRP-1 polypeptide (Accession No: P33527), R to S at positioncorresponding to position 433 of the MRP-1 polypeptide (Accession No:P33527) or R to Q at position corresponding to position 723 of the MRP-1polypeptide (Accession No: P33527). The polypeptides of encoded by thepolynucleotides of the invention have altered biological orimmunological properties due to the mutations referred to in accordancewith the present invention. Examples for said altered properties arestability of the polypeptides which may be effected or an alteredsubstrate specificity or an altered transport activity characterized by,e.g., insufficiencies in drug transport or a complete loss of thecapability of transporting drugs.

The mutations in the MRP-1 gene detected in accordance with the presentinvention are listed in Table 2. The methods of the mutation analysisfollowed standard protocols and are described in detail in the Examples.In general such methods are to be used in accordance with the presentinvention for evaluating the phenotypic spectrum as well as theoverlapping clinical characteristics of diseases or conditions relatedto dysfunctions or dysregulations and diseases related to impaired drugtransport. Advantageously, the characterization of said mutants may formthe basis of the development of improved drugs, such as drugs which areused in therapy of diseases related to multidrug resistance such as incancer therapy. Said methods encompass for example haplotype analysis,single-strand conformation polymorphism analysis (SSCA), PCR and directsequencing. On the basis of thorough clinical characterization of manypatients the phenotypes can then be correlated to these mutations aswell as to mutations that had been described earlier, for example inJounaidi, Biochem Biophys Res Commun, 221, pp. 466-470, 1996.

Also comprised by the polynucleotides referred to in the presentinvention are polynucleotides which comprise at least two of thepolynucleotides specified hereinabove, i.e. polynucleotides having anucleotide sequence which contains at least two of the mutationscomprised by the above polynucleotides or listed in Table 2 below. Thisallows the study of synergistic effects of said mutations in the MRP-1gene and/or a polypeptide encoded by said polynucleotide on thepharmacological profile of drugs in patients who bear such mutant formsof the gene or similar mutant forms that can be mimicked by the abovedescribed proteins. It is expected that the analysis of said synergisticeffects provides deeper insights into the onset of MRP-1 dysfunctions ordysregulations or diseases related to altered drug transport asdescribed supra. From said deeper insight the development of diagnosticand pharmaceutical compositions related to MRP-1 dysfunctions ordysregulations or diseases related to altered drug transport willgreatly benefit.

As is evident to the person skilled in the art, the genetic knowledgededuced from the present invention can now be used to exactly andreliably characterize the genotype of a patient. Advantageously,diseases or a prevalence for a disease which are associated with MRP-1dysfunction or dysregulation, such as cancer or other multidrugresistance related diseases referred to herein can be predicted andpreventive or therapeutical measures can be applied accordingly.Moreover in accordance with the foregoing, in cases where a given drugtakes an unusual effect, a suitable individual therapy can be designedbased on the knowledge of the individual genetic makeup of a subjectwith respect to the polynucleotides of the invention and improvedtherapeutics can be developed as will be further discussed below.

In general, the MRP-1 “status”, defined by the expression level andactivity of the MRP-1 protein, can be not only altered in many diseaseor disorders including cancer (see above), but can also be variable innormal tissue, due to genetic variations/polymorphisms. Theidentification of polymorphisms associated with altered MRP-1 expressionand/or activity is important for the prediction of drug uptake andsubsequently for the prediction of therapy outcome, including sideeffects of medications. Therefore, analysis of MRP-1 variationsindicative of MRP-1 function, is a valuable tool for therapy with drugs,which are substrates of MRP-1 and has, thanks to the present invention,now become possible.

Finally, the polynucleotides and polypeptides referred to in accordancewith the present invention are also useful as forensic markers, whichimprove the identification of subjects which have been murdered orkilled by, for example a crime of violence or any other violence and cannot be identified by the well known conventional forensic methods. Theapplication of forensic methods based on the detection of thepolymorphisms comprised by the polynucleotides of this invention in thegenome of a subject are particularly well suited in cases where a (dead)body is disfigured in a severe manner such as identification by otherbody characteristics such as the features of the face is not possible.This is the case, for example, for corpses found in water which areusually entirely disfigured. Advantageously, methods which are based onthe provision of the polynucleotides of the invention merely require aminimal amount of tissue or cells in order to be carried out. Saidtissues or cells may be blood droplets, hair roots, epidermal scales,salivia droplets, sperms etc. Since only such a minimal amount of tissueor cells is required for the identification of a subject, thepolymorphism comprised by the polynucleotides of this invention can alsobe used as forensic markers in order to proof someone guilty for acrime, such as a violation or a ravishment. Moreover, the polymorphismscomprised by the polynucleotides of this invention can be used to proofpaternity. In accordance with the forensic methods referred herein thepresence or absence of the polynucleotides of the invention isdetermined and compared with a reference sample which is unambiguouslyderived from the subject to be identified. The forensic methods whichrequire detection of the presence or absence of the polynucleotides ofthis invention in a sample of a subject the polymorphisms comprised bythe polynucleotides of this invention can be for example PCR-basedtechniques which are particularly well suited in cases where onlyminimal amount of tissue or cells is available as forensic samples. Onthe other hand, where enough tissue or cells is available, hybridizationbased techniques may be performed in order to detect the presence orabsence of a polynucleotide of this invention. These techniques are wellknown by the person skilled in the art and can be adopted to theindividual purposes referred to herein without further ado. Inconclusion, thanks to the present invention forensic means which allowimproved and reliable predictions as regards the aforementioned aspectsare now available.

In line with the foregoing, preferably, the polynucleotide of thepresent invention is associated with a disease selected from the groupof cancer diseases or multidrug resistance related diseases.

The term “cancer” used herein is very well known and characterized inthe art. Several variants of cancer exist and are comprised by said termas meant in accordance with the invention. For a detailed list ofsymptoms which are indicative for cancer it is referred to text bookknowledge, e.g. Pschyrembel.

More preferably, said cancer disease is kidney cancer, such as renalcell carcinoma (RCC). The meaning of renal cancer is explicitlydisclosed in Example 4.

In a further embodiment the present invention relates to apolynucleotide which is DNA or RNA.

The polynucleotide of the invention may be, e.g., DNA, cDNA, genomicDNA, RNA or synthetically produced DNA or RNA or a recombinantlyproduced chimeric nucleic acid molecule comprising any of thosepolynucleotides either alone or in combination. Preferably saidpolynucleotide is part of a vector, particularly plasmids, cosmids,viruses and bacteriophages used conventionally in genetic engineeringthat comprise a polynucleotide of the invention. Such vectors maycomprise further genes such as marker genes which allow for theselection of said vector in a suitable host cell and under suitableconditions.

The invention furthermore relates to a gene comprising thepolynucleotide of the invention.

It is well known in the art that genes comprise structural elementswhich encode an amino acid sequence as well as regulatory elements whichare involved in the regulation of the expression of said genes.Structural elements are represented by exons which may either encode anamino acid sequence or which may encode for RNA which is not encoding anamino acid sequence but is nevertheless involved in RNA function, e.g.by regulating the stability of the RNA or the nuclear export of the RNA.

Regulatory elements of a gene may comprise promoter elements or enhancerelements both of which could be involved in transcriptional control ofgene expression. It is very well known in the art that a promoter is tobe found upstream of the structural elements of a gene. Regulatoryelements such as enhancer elements, however, can be found distributedover the entire locus of a gene. Said elements could be reside, e.g., inintrons, regions of genomic DNA which separate the exons of a gene.Promoter or enhancer elements correspond to polynucleotide fragmentswhich are capable of attracting or binding polypeptides involved in theregulation of the gene comprising said promoter or enhancer elements.For example, polypeptides involved in regulation of said gene comprisethe so called transcription factors.

Said introns may comprise further regulatory elements which are requiredfor proper gene expression. Introns are usually transcribed togetherwith the exons of a gene resulting in a nascent RNA transcript whichcontains both, exon and intron sequences. The intron encoded RNAsequences are usually removed by a process known as RNA splicing.However, said process also requires regulatory sequences present on aRNA transcript said regulatory sequences may be encoded by the introns.

In addition, besides their function in transcriptional control andcontrol of proper RNA processing and/or stability, regulatory elementsof a gene could be also involved in the control of genetic stability ofa gene locus. Said elements control, e.g., recombination events or serveto maintain a certain structure of the DNA or the arrangement of DNA ina chromosome.

Therefore, single nucleotide polymorphisms can occur in exons of a genewhich encode an amino acid sequence as discussed supra as well as inregulatory regions which are involved in the above discussed process.The analysis of the nucleotide sequence of a gene locus in its entiretyincluding, e.g., introns is in light of the above desirable. Thepolymorphisms comprised by the polynucleotides of the present inventioncan influence the expression level of MRP-1 protein via mechanismsinvolving enhanced or reduced transcription of the MRP-1 gene,stabilization of the gene's RNA transcripts and alteration of theprocessing of the primary RNA transcripts.

Therefore, in a furthermore preferred embodiment of the gene of theinvention a nucleotide deletion, addition and/or substitution results inaltered expression of the variant gene compared to the correspondingwild type gene.

In another embodiment the present invention relates to a vectorcomprising the polynucleotide of the invention or the gene of theinvention.

Said vector may be, for example, a phage, plasmid, viral or retroviralvector. Retroviral vectors may be replication competent or replicationdefective. In the latter case, viral propagation generally will occuronly in complementing host/cells.

The polynucleotides or genes of the invention may be joined to a vectorcontaining selectable markers for propagation in a host. Generally, aplasmid: vector is introduced in a precipitate such as a calciumphosphate precipitate, or in a complex with a charged lipid or incarbon-based clusters. Should the vector be a virus, it may be packagedin vitro using an appropriate packaging cell line prior to applicationto host cells.

In a more preferred embodiment of the vector of the invention thepolynucleotide is operatively linked to expression control sequencesallowing expression in prokaryotic or eukaryotic cells or isolatedfractions thereof.

Expression of said polynucleotide comprises transcription of thepolynucleotide, preferably into a translatable mRNA. Regulatory elementsensuring expression in eukaryotic cells, preferably mammalian cells, arewell known to those skilled in the art. They usually comprise regulatorysequences ensuring initiation of transcription and optionally poly-Asignals ensuring termination of transcription and stabilization of thetranscript. Additional regulatory elements may include transcriptionalas well as translational enhancers. Possible regulatory elementspermitting expression in prokaryotic host cells comprise, e.g., the lac,trp or tac promoter in E. coli, and examples for regulatory elementspermitting expression in eukaryotic host cells are the AOX1 or GAL1promoter in yeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus),CMV-enhancer, SV40-enhancer or a globin intron in mammalian and otheranimal cells. Beside elements which are responsible for the initiationof transcription such regulatory elements may also comprisetranscription termination signals, such as the SV40-poly-A site or thetk-poly-A site, downstream of the polynucleotide. In this context,suitable expression vectors are known in the art such as Okayama-BergcDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3(In-vitrogene), pSPORT1 (GIBCO BRL). Preferably, said vector is anexpression vector and/or a gene transfer or targeting vector. Expressionvectors derived from viruses such as retroviruses, vaccinia virus,adeno-associated virus, herpes viruses, or bovine papilloma virus, maybe used for delivery of the polynucleotides or vector of the inventioninto targeted cell population. Methods which are well known to thoseskilled in the art can be used to construct recombinant viral vectors;see, for example, the techniques described in Sambrook, MolecularCloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.and Ausubel, Current Protocols in Molecular Biology, Green PublishingAssociates and Wiley Interscience, N.Y. (1994). Alternatively, thepolynucleotides and vectors of the invention can be reconstituted intoliposomes for delivery to target cells.

The term “isolated fractions thereof” refers to fractions of eukaryoticor prokaryotic cells or tissues which are capable of transcribing ortranscribing and translating RNA from the vector of the invention. Saidfractions comprise proteins which are required for transcription of RNAor transcription of RNA and translation of said RNA into a polypeptide.Said isolated fractions may be, e.g., nuclear and cytoplasmic fractionsof eukaryotic cells such as of reticulocytes.

The present invention furthermore relates to a host cell geneticallyengineered with the polynucleotide of the invention, the gene of theinvention or the vector of the invention.

Said host cell may be a prokaryotic or eukaryotic cell; see supra. Thepolynucleotide or vector of the invention which is present in the hostcell may either be integrated into the genome of the host cell or it maybe maintained extrachromosomally. In this respect, it is also to beunderstood that the recombinant DNA molecule of the invention can beused for “gene targeting” and/or “gene replacement”, for restoring amutant gene or for creating a mutant gene via homologous recombination;see for example Mouellic, Proc. Natl. Acad. Sci. USA, 87 (1990),4712-4716; Joyner, Gene Targeting, A Practical Approach, OxfordUniversity Press.

The host cell can be any prokaryotic or eukaryotic cell, such as abacterial, insect, fungal, plant, animal, mammalian or, preferably,human cell. Preferred fungal cells are, for example, those of the genusSaccharomyces, in particular those of the species S. cerevisiae. Theterm “prokaryotic” is meant to include all bacteria which can betransformed or transfected with a polynucleotide for the expression of avariant polypeptide of the invention. Prokaryotic hosts may include gramnegative as well as gram positive bacteria such as, for example, E.coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. Apolynucleotide coding for a mutant form of variant polypeptides of theinvention can be used to transform or transfect the host using any ofthe techniques commonly known to those of ordinary skill in the art.Methods for preparing fused, operably linked genes and expressing themin bacteria or animal cells are well-known in the art (Sambrook, supra).The genetic constructs and methods described therein can be utilized forexpression of variant polypeptides of the invention in, e.g.,prokaryotic hosts. In general, expression vectors containing promotersequences which facilitate the efficient transcription of the insertedpolynucleotide are used in connection with the host. The expressionvector typically contains an origin of replication, a promoter, and aterminator, as well as specific genes which are capable of providingphenotypic selection of the transformed cells. The transformedprokaryotic hosts can be grown in fermentors and cultured according totechniques known in the art to achieve optimal cell growth. The proteinsof the invention can then be isolated from the grown medium, cellularlysates, or cellular membrane fractions. The isolation and purificationof the microbially or otherwise expressed polypeptides of the inventionmay be by any conventional means such as, for example, preparativechromatographic separations and immunological separations such as thoseinvolving the use of monoclonal or polyclonal antibodies.

Thus, in a further embodiment the invention relates to a method forproducing a molecular variant MRP-1 polypeptide or fragment thereofcomprising culturing the above described host cell; and recovering saidprotein or fragment from the culture.

In another embodiment the present invention relates to a method forproducing cells capable of expressing a molecular variant MRP-1polypeptide comprising genetically engineering cells with thepolynucleotide of the invention, the gene of the invention or the vectorof the invention.

The cells obtainable by the method of the invention can be used, forexample, to test drugs according to the methods described in D. L.Spector, R. D. Goldman, L. A. Leinwand, Cells, a Lab manual, CSH Press1998. Furthermore, the cells can be used to study known drugs andunknown derivatives thereof for their ability to complement thedeficiency caused by mutations in the MRP-1 gene. For these embodimentsthe host cells preferably lack a wild type allele, preferably bothalleles of the MRP-1 gene and/or have at least one mutated from thereof.Ideally, the gene comprising an allele as comprised by thepolynucleotides of the invention could be introduced into the wild typelocus by homologous replacement. Alternatively, strong overexpression ofa mutated allele over the normal allele and comparison with arecombinant cell line overexpressing the normal allele at a similarlevel may be used as a screening and analysis system. The cellsobtainable by the above-described method may also be used for thescreening methods referred to herein below.

Furthermore, the invention relates to a polypeptide or fragment thereofencoded by the polynucleotide of the invention, the gene of theinvention or obtainable by the method described above or from cellsproduced by the method described above. In this context it is alsounderstood that the variant polypeptide of the invention can be furthermodified by conventional methods known in the art. By providing saidvariant proteins according to the present invention it is also possibleto determine the portions relevant for their biological activity orinhibition of the same. The terms “polypeptide” and “protein” as usedherein are exchangeable. Moreover, what is comprised by said terms isstandard textbook knowledge.

The present invention furthermore relates to an antibody which bindsspecifically to the polypeptide of the invention.

Advantageously, the antibody specifically recognizes or binds an epitopecontaining one or more amino acid substitution(s) as defined above.Antibodies against the variant polypeptides of the invention can beprepared by well known methods using a purified protein according to theinvention or a (synthetic) fragment derived therefrom as an antigen.Monoclonal antibodies can be prepared, for example, by the techniques asoriginally described in Köhler and Milstein, Nature 256 (1975), 495, andGalfré, Meth. Enzymol. 73 (1981), 3, which comprise the fusion of mousemyeloma cells to spleen cells derived from immunized mammals. In apreferred embodiment of the invention, said antibody is a monoclonalantibody, a polyclonal antibody, a single chain antibody, human orhumanized antibody, primatized, chimerized or fragment thereof thatspecifically binds said peptide or polypeptide also including bispecificantibody, synthetic antibody, antibody fragment, such as Fab, Fv or scFvfragments etc., or a chemically modified derivative of any of these.Furthermore, antibodies or fragments thereof to the aforementionedpolypeptides can be obtained by using methods which are described, e.g.,in Harlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, ColdSpring Harbor, 1988. These antibodies can be used, for example, for theimmunoprecipitation and immunolocalization of the variant polypeptidesof the invention as well as for the monitoring of the presence of saidvariant polypeptides, for example, in recombinant organisms, and for theidentification of compounds interacting with the proteins according tothe invention. For example, surface plasmon resonance as employed in theBIAcore system can be used to increase the efficiency of phageantibodies which bind to an epitope of the protein of the invention(Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J.Immunol. Methods 183 (1995), 7-13).

In a preferred embodiment the antibody of the present inventionspecifically recognizes an epitope containing one or more amino acidsubstitution(s) resulting from a nucleotide exchange as defined supra.

Antibodies which specifically recognize modified amino acids such asphospho-Tyrosine residues are well known in the art. Similarly, inaccordance with the present invention antibodies which specificallyrecognize even a single amino acid exchange in an epitope may begenerated by the well known methods described supra.

In light of the foregoing, in a more preferred embodiment the antibodyof the present invention is monoclonal or polyclonal.

The invention also relates to a transgenic non-human animal comprisingat least one polynucleotide of the invention, the gene of the inventionor the vector of the invention as described supra.

The present invention also encompasses a method for the production of atransgenic non-human animal comprising introduction of a polynucleotideor vector of the invention into a germ cell, an embryonic cell, stemcell or an egg or a cell derived therefrom. The non-human animal can beused in accordance with the method of the invention described below andmay be a non-transgenic healthy animal, or may have a disease ordisorder, preferably a disease caused by at least one mutation in thegene of the invention. Such transgenic animals are well suited for,e.g., pharmacological studies of drugs in connection with variant formsof the above described variant polypeptides since these polypeptides orat least their functional domains are conserved between species inhigher eukaryotes, particularly in mammals. Production of transgenicembryos and screening of those can be performed, e.g., as described byA. L. Joyner Ed., Gene Targeting, A Practical Approach (1993), OxfordUniversity Press. The DNA of the embryos can be analyzed using, e.g.,Southern blots with an appropriate probe or based on PCR techniques. Atransgenic non-human animal in accordance with the invention may be atransgenic mouse, rat, hamster, dog, monkey, rabbit, pig, frog, nematodesuch as Caenorhabditis elegans, fruitfly such as Drosophila melanogasteror fish such as torpedo fish or zebrafish comprising a polynucleotide orvector of the invention or obtained by the method described above,preferably wherein said polynucleotide or vector is stably integratedinto the genome of said non-human animal, preferably such that thepresence of said polynucleotide or vector leads to the expression of thevariant polypeptide of the invention. It may comprise one or severalcopies of the same or different polynucleotides or genes of theinvention. This animal has numerous utilities, including as a researchmodel for cardiovascular research and therefore, presents a novel andvaluable animal in the development of therapies, treatment, etc. fordiseases caused by cardiovascular diseases. Accordingly, in thisinstance, the mammal is preferably a laboratory animal such as a mouseor rat.

Thus, in a preferred embodiment the transgenic non-human animal of theinvention is a mouse, a rat or a zebrafish.

Numerous reports revealed that said animals are particularly well suitedas model organisms for the investigation of the drug metabolism and itsdeficiencies or cancer. Advantageously, transgenic animals can be easilycreated using said model organisms, due to the availability of varioussuitable techniques well known in the art.

The invention also relates to a solid support comprising one or aplurality of the polynucleotide, the gene, the vector, the polypeptide,the antibody or the host cell of the invention in immobilized form.

The term “solid support” as used herein refers to a flexible ornon-flexible support that is suitable for carrying said immobilizedtargets. Said solid support may be homogenous or inhomogeneous. Forexample, said solid support may consist of different materials havingthe same or different properties with respect to flexibility andimmobilization, for instance, or said solid support may consist of onematerial exhibiting a plurality of properties also comprisingflexibility and immobilization properties. Said solid support maycomprise glass-, polypropylene- or silicon-chips, membranesoligonucleotide-conjugated beads or bead arrays.

The term “immobilized” means that the molecular species of interest isfixed to a solid support, preferably covalently linked thereto. Thiscovalent linkage can be achieved by different means depending on themolecular nature of the molecular species. Moreover, the molecularspecies may be also fixed on the solid support by electrostatic forces,hydrophobic or hydrophilic interactions or Van-der-Waals forces. Theabove described physico-chemical interactions typically occur ininteractions between molecules. For example, biotinylated polypeptidesmay be fixed on a avidin-coated solid support due to interactions of theabove described types. Further, polypeptides such as antibodies, may befixed on an antibody coated solid support. Moreover, the immobilizationis dependent on the chemical properties of the solid support. Forexample, the nucleic acid molecules can be immobilized on a membrane bystandard techniques such as UV-crosslinking or heat.

In a preferred embodiment of the invention said solid support is amembrane, a glass- or poylpropylene- or silicon-chip, are membranesoligonucleotide-conjugated beads or a bead array, which is assembled onan optical filter substrate.

Moreover, the present invention relates to an in vitro method foridentifying a polymorphism said method comprising the steps of:

-   (a) isolating a polynucleotide or the gene of the invention from a    plurality of subgroups of individuals, wherein one subgroup has no    prevalence for a MRP-1 associated disease and at least one or more    further subgroup(s) do have prevalence for a MRP-1 associated    disease; and-   (b) identifying a polymorphism by comparing the nucleic acid    sequence of said polynucleotide or said gene of said one subgroup    having no prevalence for a MRP-1 associated disease with said at    least one or more further subgroup(s) having a prevalence for a    MRP-1 associated disease.

The term “prevalence” as used herein means that individuals are besusceptible for one or more disease(s) which are associated with MRP-1dysfunction or dysregulation or could already have one or more of saiddisease(s). Thereby, one MRP-1 associated disease can be used todetermine the susceptibility for another MRP-1 associated disease, e.g.altered drug transport may be indicative for a prevalence for, e.g.cancer. Moreover, symptoms which are indicative for a prevalence fordeveloping said diseases are very well known in the art and have beensufficiently described in standard textbooks such as Pschyrembel.

Advantageously, polymorphisms according to the present invention whichare associated with MRP-1 dysfunction or dysregulation or one or moredisease(s) based thereon should be enriched in subgroups of individualswhich have a prevalence for said diseases versus subgroups which have noprevalence for said diseases. Thus, the above described method allowsthe rapid and reliable detection of polymorphism which are indicativefor one or more MRP-1 associated disease(s) or a susceptibilitytherefor. Advantageously, due to the phenotypic preselection a largenumber of individuals having no prevalence might be screened forpolymorphisms in general. Thereby, a reference sequences comprisingpolymorphisms which do not correlate to one or more MRP-1 associateddisease(s) can be obtained. Based on said reference sequences it ispossible to efficiently and reliably determine the relevantpolymorphisms.

In a further embodiment the present invention relates to a method foridentifying and obtaining a pro-drug or a drug capable of modulating theactivity of a molecular variant of a MRP-1 polypeptide comprising thesteps of:

-   (a) contacting the polypeptide, the solid support of the invention,    a cell expressing a molecular variant gene comprising a    polynucleotide of the invention, the gene or the vector of the    invention in the presence of components capable of providing a    detectable signal in response to drug activity with a compound to be    screened for pro-drug or drug activity; and-   (b) detecting the presence or absence of a signal or increase or    decrease of a signal generated from the pro-drug or the drug    activity, wherein the absence, presence, increase or decrease of the    signal is indicative for a putative pro-drug or drug.

The term “compound” in a method of the invention includes a singlesubstance or a plurality of substances which may or may not beidentical.

Said compound(s) may be chemically synthesized or produced via microbialfermentation but can also be comprised in, for example, samples, e.g.,cell extracts from, e.g., plants, animals or microorganisms.Furthermore, said compounds may be known in the art but hitherto notknown to be useful as an inhibitor, respectively. The plurality ofcompounds may be, e.g., added to the culture medium or injected into acell or non-human animal of the invention.

If a sample containing (a) compound(s) is identified in the method ofthe invention, then it is either possible to isolate the compound fromthe original sample identified as containing the compound, in questionor one can further subdivide the original sample, for example, if itconsists of a plurality of different compounds, so as to reduce thenumber of different substances per sample and repeat the method with thesubdivisions of the original sample. It can then be determined whethersaid sample or compound displays the desired properties, for example, bythe methods described herein or in the literature (Spector et al., Cellsmanual; see supra). Depending on the complexity of the samples, thesteps described above can be performed several times, preferably untilthe sample identified according to the method of the invention onlycomprises a limited number of or only one substance(s). Preferably saidsample comprises substances of similar chemical and/or physicalproperties, and most preferably said substances are identical. Themethods of the present invention can be easily performed and designed bythe person skilled in the art, for example in accordance with other cellbased assays described in the prior art or by using and modifying themethods as described herein. Furthermore, the person skilled in the artwill readily recognize which further compounds may be used in order toperform the methods of the invention, for example, enzymes, ifnecessary, that convert a certain compound into a precursor. Suchadaptation of the method of the invention is well within the skill ofthe person skilled in the art and can be performed without undueexperimentation.

Compounds which can be used in accordance with the present inventioninclude peptides, proteins, nucleic acids, antibodies, small organiccompounds, ligands, peptidomimetics, PNAs and the like. Said compoundsmay act as agonists or antagonists of the invention. Said compounds canalso be functional derivatives or analogues of known drugs. Methods forthe preparation of chemical derivatives and analogues are well known tothose skilled in the art and are described in, for example, Beilstein,Handbook of Organic Chemistry, Springer edition New York Inc., 175 FifthAvenue, New York, N.Y. 10010 U.S.A. and Organic Synthesis, Wiley, NewYork, USA. Furthermore, said derivatives and analogues can be tested fortheir effects according to methods known in the art or as described.Furthermore, peptide mimetics and/or computer aided design ofappropriate drug derivatives and analogues can be used, for example,according to the methods described below. Such analogs comprisemolecules may have as the basis structure of known MRP-1 substratesand/or inhibitors and/or modulators; see infra.

Appropriate computer programs can be used for the identification ofinteractive sites of a putative inhibitor and the polypeptides of theinvention by computer assistant searches for complementary structuralmotifs (Fassina, Immunomethods 5 (1994), 114-120). Further appropriatecomputer systems for the computer aided design of protein and peptidesare described in the prior art, for example, in Berry, Biochem, Soc.:Trans. 22 (1994), 1033-1036; Wodak, Ann. N.Y. Acad. Sci. 501 (1987),1-13; Pabo, Biochemistry 25 (1986), 5987-5991. The results obtained fromthe above-described computer analysis can be used in combination withthe method of the invention for, e.g., optimizing known inhibitors,analogs, antagonists or agonists. Appropriate peptidomimetics and otherinhibitors can also be identified by the synthesis of peptidomimeticcombinatorial libraries through successive chemical modification andtesting the resulting compounds, e.g., according to the methodsdescribed herein. Methods for the generation and use of peptidomimeticcombinatorial libraries are described in the prior art, for example inOstresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg.Med. Chem. 4 (1996), 709-715. Furthermore, the three-dimensional and/orcrystallographic structure of said compounds and the polypeptides of theinvention can be used for the design of peptidomimetic drugs (Rose,Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4(1996), 1545-1558). It is very well known how to obtain said compounds,e.g. by chemical or biochemical standard techniques. Thus, alsocomprised by the method of the invention are means of making orproducing said compounds. In summary, the present invention providesmethods for identifying and obtaining compounds which can be used inspecific doses for the treatment of specific forms of MRP-1 associateddiseases, e.g. dysfunctions or dysregulations of the drug transport suchas cancer or multidrug resistance.

The above definitions apply mutatis mutandis to all of the methodsdescribed in the following.

In a further embodiment the present invention relates to a method foridentifying and obtaining an inhibitor of the activity of a molecularvariant of a MRP-1 polypeptide comprising the steps of:

-   (a) contacting the protein, the solid support of the invention or a    cell expressing a molecular variant gene comprising a polynucleotide    or the gene or the vector of the invention in the presence of    components capable of providing a detectable signal in response to    drug activity with a compound to be screened for inhibiting    activity; and-   (b) detecting the presence or absence of a signal or increase or    decrease of a signal generated from the inhibiting activity, wherein    the absence or decrease of the signal is indicative for a putative    inhibitor.

In a preferred embodiment of the method of the invention said cell is acell, obtained by the method of the invention or can be obtained fromthe transgenic non-human animal as described supra.

In a still further embodiment the present invention relates to a methodof identifying and obtaining a pro-drug or drug capable of modulatingthe activity of a molecular variant of a MRP-1 polypeptide comprisingthe steps of:

-   (a) contacting the host cell, the cell obtained by the method of the    invention, the polypeptide or the solid support of the invention    with the first molecule known to be bound by a MRP-1 polypeptide to    form a first complex of said polypeptide and said first molecule;-   (b) contacting said first complex with a compound to be screened,    and-   (c) measuring whether said compound displaces said first molecule    from said first complex.

Advantageously, in said method said measuring step comprises measuringthe formation of a second complex of said protein and said inhibitorcandidate. Preferably, said measuring step comprises measuring theamount of said first molecule that is not bound to said protein.

In a particularly preferred embodiment of the above-described method ofsaid first molecule is a agonist or antagonist or a substrate and/or ainhibitor and/or a modulator of the polypeptide of the invention, e.g.,with a radioactive or fluorescent label.

In a still another embodiment the present invention relates to a methodof identifying and obtaining an inhibitor capable of modulating theactivity of a molecular variant of a MRP-1 polypeptide comprising thesteps of:

-   (a) contacting the host cell or the cell obtained by the method of    the invention, the protein or the solid support of the invention    with the first molecule known to be bound by the MRP-1 polypeptide    to form a first complex of said protein and said first molecule;-   (b) contacting said first complex with a compound to be screened,    and-   (c) measuring whether said compound displaces said first molecule    from said first complex.

In a preferred embodiment of the method of the invention said measuringstep comprises measuring the formation of a second complex of saidprotein and said compound.

In another preferred embodiment of the method of the invention saidmeasuring step comprises measuring the amount of said first moleculethat is not bound to said protein.

In a more preferred embodiment of the method of the invention said firstmolecule is labeled.

The invention furthermore relates to a method for the production of apharmaceutical composition comprising the steps of the method asdescribed supra; and the further step of formulating the compoundidentified and obtained or a derivative thereof in a pharmaceuticallyacceptable form.

The therapeutically useful compounds identified according to the methodsof the invention can be formulated and administered to a patient asdiscussed above. For uses and therapeutic doses determined to beappropriate by one skilled in the art and for definitions of the term“pharmaceutical composition” see infra.

Furthermore, the present invention encompasses a method for thepreparation of a pharmaceutical composition comprising the steps of theabove-described methods; and formulating a drug or pro-drug in the formsuitable for therapeutic application and preventing or ameliorating thedisorder of the subject diagnosed in the method of the invention.

Drugs or pro-drugs after their in vivo administration are metabolized inorder to be eliminated either by excretion or by metabolism to one ormore active or inactive metabolites (Meyer, J. Pharmacokinet. Biopharm.24 (1996), 449-459). Thus, rather than using the actual compound orinhibitor identified and obtained in accordance with the methods of thepresent invention a corresponding formulation as a pro-drug can be usedwhich is converted into its active in the patient. Precautionarymeasures that may be taken for the application of pro-drugs and drugsare described in the literature; see, for review, Ozama, J. Toxicol.Sci. 21 (1996), 323-329).

In a preferred embodiment of the method of the present invention saiddrug or prodrug is a derivative of a medicament as defined hereinafter.

The present invention also relates to a method of diagnosing a disorderrelated to the presence of a molecular variant of the MRP-1 gene orsusceptibility to such a disorder comprising determining the presence ofa polynucleotide or the gene of the invention in a sample from asubject.

In accordance with this embodiment of the present invention, the methodof testing the status of a disorder or susceptibility to such a disordercan be effected by using a polynucleotide gene or nucleic acid of theinvention, e.g., in the form of a Southern or Northern blot or in situanalysis. Said nucleic acid sequence may hybridize to a coding region ofeither of the genes or to a non-coding region, e.g. intron. In the casethat a complementary sequence is employed in the method of theinvention, said nucleic acid molecule can again be used in Northernblots. Additionally, said testing can be done in conjunction with anactual blocking, e.g., of the transcription of the gene and thus isexpected to have therapeutic relevance. Furthermore, a primer oroligonucleotide can also be used for hybridizing to one of the abovementioned MRP-1 gene or corresponding mRNAs. The nucleic acids used forhybridization can, of course, be conveniently labeled by incorporatingor attaching, e.g., a radioactive or other marker. Such markers are wellknown in the art. The labeling of said nucleic acid molecules can beeffected by conventional methods.

Additionally, the presence or expression of variant MRP-1 gene can bemonitored by using a primer pair that specifically hybridizes to eitherof the corresponding nucleic acid sequences and by carrying out a PCRreaction according to standard procedures. Specific hybridization of theabove mentioned probes or primers preferably occurs at stringenthybridization conditions. The term “stringent hybridization conditions”is well known in the art; see, for example, Sambrook et al., “MolecularCloning, A Laboratory Manual” second ed., CSH Press, Cold Spring Harbor,1989; “Nucleic Acid Hybridisation, A Practical Approach”, Hames andHiggins eds., IRL Press, Oxford, 1985. Furthermore, the mRNA, cRNA, cDNAor genomic DNA obtained from the subject may be sequenced to identifymutations which may be characteristic fingerprints of mutations in thepolynucleotide or the gene of the invention. The present inventionfurther comprises methods wherein such a fingerprint may be generated byRFLPs of DNA or RNA obtained from the subject, optionally the DNA or RNAmay be amplified prior to analysis, the methods of which are well knownin the art. RNA fingerprints may be performed by, for example, digestingan RNA sample obtained from the subject with a suitable RNA-Enzyme, forexample RNase T₁, RNase T₂ or the like or a ribozyme and, for example,electrophoretically separating and detecting the RNA fragments asdescribed above. Further modifications of the above-mentioned embodimentof the invention can be easily devised by the person skilled in the art,without any undue experimentation from this disclosure; see, e.g., theexamples. An additional embodiment of the present invention relates to amethod wherein said determination is effected by employing an antibodyof the invention or fragment thereof. The antibody used in the method ofthe invention may be labeled with detectable tags such as a histidineflags or a biotin molecule.

The invention relates to a method of diagnosing a disorder related tothe presence of a molecular variant of a MRP-1 gene or susceptibility tosuch a disorder comprising determining the presence of a polypeptide orthe antibody of the invention in a sample from a subject.

In a preferred embodiment of the above described method said disorder isa cancer disease or a disease related to multidrug resistance.

In a preferred embodiment of the present invention, the above describedmethod is comprising PCR, ligase chain reaction, restriction digestion,direct sequencing, nucleic acid amplification techniques, hybridizationtechniques or immunoassays. Said techniques are very well known in theart.

Moreover, the invention relates to a method of detection of thepolynucleotide or the gene of the invention in a sample comprising thesteps of

-   -   (a) contacting the solid support described supra with the sample        under conditions allowing interaction of the polynucleotide or        the gene of the invention with the immobilized targets on a        solid support and;

-   (b) determining the binding of said polynucleotide or said gene to    said immobilized targets on a solid support.

The invention also relates to an in vitro method for diagnosing adisease comprising the steps of the method described supra, whereinbinding of said polynucleotide or gene to said immobilized targets onsaid solid support is indicative for the presence or the absence of saiddisease or a prevalence for said disease.

The invention furthermore relates to a diagnostic composition comprisingthe polynucleotide, the gene, the vector, the polypeptide or theantibody of the invention.

In addition, the invention relates to a pharmaceutical compositioncomprising the polynucleotide, the gene, the vector, the polypeptide orthe antibody of the invention. These pharmaceutical compositionscomprising, e.g., the antibody may conveniently be administered by anyof the routes conventionally used for drug administration, for instance,orally, topically, parenterally or by inhalation. Acceptable saltscomprise acetate, methylester, HCl, sulfate, chloride and the like. Thecompounds may be administered in conventional dosage forms prepared bycombining the drugs with standard pharmaceutical carriers according toconventional procedures. These procedures may involve mixing,granulating and compressing or dissolving the ingredients as appropriateto the desired preparation. It will be appreciated that the form andcharacter of the pharmaceutically acceptable character or diluent isdictated by the amount of active ingredient with which it is to becombined, the route of administration and other well-known variables.The carrier(s) must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not deleterious to therecipient thereof. The pharmaceutical carrier employed may be, forexample, either a solid or liquid. Exemplary of solid carriers arelactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia,magnesium stearate, stearic acid and the like. Exemplary of liquidcarriers are phosphate buffered saline solution, syrup, oil such aspeanut oil and olive oil, water, emulsions, various types of wettingagents, sterile solutions and the like. Similarly, the carrier ordiluent may include time delay material well known to the art, such asglyceryl mono-stearate or glyceryl distearate alone or with a wax.

The dosage regimen will be determined by the attending physician andother clinical factors; preferably in accordance with any one of theabove described methods. As is well known in the medical arts, dosagesfor any one patient depends upon many factors, including the patient'ssize, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. Progress can be monitoredby periodic assessment.

Furthermore, the use of pharmaceutical compositions which compriseantisense-oligonucleotides which specifically hybridize to RNA encodingmutated versions of the polynucleotide or gene according to theinvention or which comprise antibodies specifically recognizing amutated polypeptide of the invention but not or not substantially thefunctional wild-type form is conceivable in cases in which theconcentration of the mutated form in the cells should be reduced.

Thanks to the present invention the particular drug selection, dosageregimen and corresponding patients to be treated can be determined inaccordance with the present invention. The dosing recommendations willbe indicated in product labeling by allowing the prescriber toanticipate dose adjustments depending on the considered patient group,with information that avoids prescribing the wrong drug to the wrongpatients at the wrong dose.

In another embodiment the present invention relates to the use of thepolynucleotide, the gene, the vector, the polypeptide thepolynucleotides having at a position corresponding to position 926 ofthe MRP-1 gene (Accession No: U07050) a T insertion, at a positioncorresponding to position 79 of the MRP-1 gene (Accession No: AF022830)an A or at a position corresponding to position 137647 of the MRP-1 gene(Accession No: AC026452) a T, or at a position corresponding to position150727 of the MRP-1 gene (Accession No: AC025277) an A, or the antibodyof the invention for the preparation of a diagnostic composition fordiagnosing a disease. A gene encoding a functional and expressiblepolypeptide of the invention can be introduced into the cells which inturn produce the protein of interest. Gene therapy, which is based onintroducing therapeutic genes into cells by ex-vivo or in-vivotechniques is one of the most important applications of gene transfer.Suitable vectors and methods for in-vitro or in-vivo gene therapy aredescribed in the literature and are known to the person skilled in theart; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper,Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813;Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995),1077-1086; Wang, Nature Medicine 2 (1996), 714-716; WO94/29469; WO97/00957 or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640,and references cited therein. The gene may be designed for directintroduction or for introduction via liposomes, or viral vectors (e.g.adenoviral, retroviral) into the cell. Preferably, said cell is a germline cell, embryonic cell, or egg cell or derived therefrom, mostpreferably said cell is a stem cell.

As is evident from the above, it is preferred that in the use of theinvention the nucleic acid sequence is operatively linked to regulatoryelements allowing for the expression and/or targeting of thepolypeptides of the invention to specific cells. Suitable gene deliverysystems that can be employed in accordance with the invention mayinclude liposomes, receptor-mediated delivery systems, naked DNA, andviral vectors such as herpes viruses, retroviruses, adenoviruses, andadeno-associated viruses, among others. Delivery of nucleic acids to aspecific site in the body for gene therapy may also be accomplishedusing a biolistic delivery system, such as that described by Williams(Proc. Natl. Acad. Sci. USA 88 (1991), 2726-2729). Standard methods fortransfecting cells with recombinant DNA are well known to those skilledin the art of molecular biology, see, e.g., WO 94/29469; see also supra.Gene therapy may be carried out by directly administering therecombinant DNA molecule or vector of the invention to a patient or bytransfecting cells with the polynucleotide or vector of the invention exvivo and infusing the transfected cells into the patient.

In a further embodiment the present invention relates to the use of thepolynucleotide, the gene, the vector, the polypeptide thepolynucleotides having at a position corresponding to position 926 ofthe MRP-1 gene (Accession No: U07050) a T insertion, at a positioncorresponding to position 79 of the MRP-1 gene (Accession No: AF022830)an A or at a position corresponding to position 137647 of the MRP-1 gene(Accession No: AC026452) a T, or at a position corresponding to position150727 of the MRP-1 gene (Accession No: AC025277) an A, or the antibodyof the invention for the preparation of a pharmaceutical composition fortreating a disease.

In a more preferred embodiment of the use of the present invention saiddisease is cancer or a disease related to multidrug resistance.

Finally, the present invention relates to a diagnostic kit for detectionof a single nucleotide polymorphism comprising the polynucleotide, thegene, the vector, the polypeptide, the antibody, the host cell, thetransgenic non-human animal or the solid support of the invention.

The kit of the invention may contain further ingredients such asselection markers and components for selective media suitable for thegeneration of transgenic cells and animals. The kit of the invention canbe used for carrying out a method of the invention and could be, interalia, employed in a variety of applications, e.g., in the diagnosticfield or as research tool. The parts of the kit of the invention can bepackaged individually in vials or other appropriate means depending onthe respective ingredient or in combination in suitable containers ormulticontainer units. Manufacture of the kit follows preferably standardprocedures which are known to the person skilled in the art. The kit maybe used for methods for detecting expression of a mutant form of thepolypeptides, genes or polynucleotides in accordance with any one of theabove-described methods of the invention, employing, for example,immunoassay techniques such as radioimmunoassay or enzymeimmunoassay orpreferably nucleic acid hybridization and/or amplification techniquessuch as those described herein before and in the Examples as well aspharmacokinetic studies when using non-human transgenic animals of theinvention.

The figures illustrate the invention:

FIG. 1: The figure shows, where the novel MRP-1 SNP's are located on thegene and the protein, respectively.

FIG. 2: The figure illustrates the correlation between MRP-1 transportactivity, intracellular carcinogen/drug concentrations and cancer risk,therapy outcome and side effects.

FIG. 3: Diagram 1A and 1B represent the correlation of the genotype(wt/wt: 1; wt/mut and mut/mut:2) with MRP-1 mRNA content in duodenalbiopsies from healthy volunteers derived from two independentexperiments, before (A) and after (B) application of rifampicin. Thep-value of the statistical evaluation (Kruskal-Wallis-Test), whichresult in a genotyp/phenotype correlation is p=0.086. The p-value of thepaired T-test (p<0.001) demonstrates, that rifampicin has no effect onMRP-1 mRNA expression. Thus, the differences in the MRP-1 mRNA contentare based on interindividual differences. The statistical analyses wereperformed using the computer program SPSS 10.0 (SPSS, Chicago, USA).

The invention will now be described by reference to the followingbiological Examples which are merely illustrative and are notconstructed as a limitation of the scope of the present invention.

EXAMPLE 1 Isolation of Genomic DNA from Human Blood, Generation andPurification of MRP-1 fragments

Genomic DNA was obtained by standard ion exchange chromatographytechniques (Quiagen kits for isolation of genomic DNA from blood).Specific oligonucleotide primers, 2 for each fragment, were applied toobtain defined DNA fragments by polymerase chain reaction (PCR)containing specific parts of the MRP-1 gene. These specificoligonucleotide primers were designed to bind to sequences upstream anddownstream of various exons of the gene. The resulting DNA fragmentswere to encode not only exon sequences, but also some intron sequencesat the exon-intron boundaries. Such intronic sequences adjacent to theexons are known to be important for correct splicing and subsequentexpression of the mRNA, which encodes for the respective protein.Oligonucleotide primer pairs that were optimized for each of the PCRfragments, synthesized and purified by affinity chromatography (OPCcartridges). The primer sequences for the amplification of the singlefragments are listed in Table 1.

Polymerase chain reactions for the single MRP-1 gene fragments, wereperformed under conditions, that were optimized for each of thesefragments. These MRP-1 gene fragments cover the respective exons, aswell as regulatory regions, like promoter, 5′-UTR and 3′-UTR (see Table1). PCRs were carried out for all fragments in a reaction volume of 50μl. 40 ng DNA template was added to standard PCR buffer containing 1.5mM MgCl2 (Qiagen, Hilden), 200 μM dNTP's (Roth, Karlsruhe), 0.4 μM(conditions A and C) or 1.6 μM (condition B) of each primer (Metabion,Munich), 10 μl Q-Solution (condition C; Qiagen, Hilden), 4 μl DMSO(condition B) and 1 U Taq polymerase (Qiagen, Hilden). All PCRs(conditions A and C) were performed on a Perkin Elmer thermocycler(model 9700) with an initial denaturation step of 2 min at 94° C. and 34amplification cycles of denaturation at 94° C. for 45 sec, primerannealing at 62° C. for 45 sec, and 1 min for 72° C. followed by a finalextension of 72° C. for 10 min. In the case of condition B the PCRreaction was performed with an initial denaturation step of 3 min at 96°C. and 35 amplification cycles of denaturation at 96° C. for 45 sec,primer annealing at 62° C. for 30 sec, and 1 min for 72° C. followed bya final extension of 72° C. for 10 min.

The optimized PCR-conditions and the resulting size of the desired andobtained fragments are listed in Table 1. The defined DNA fragmentscontaining specific parts of the human MRP-1 gene were processed toremove nonincorporated nucleotides and buffer components that otherwiseinterfere with the subsequent determination of the individual MRP-1genotype by direct DNA sequencing. For this purification, standard ionexchange chromatography techniques were used (Quiagen kits for PCRfragment purification). For all of the fragments, sufficient yields ofpurified fragments, suitable for direct DNA sequence analyses, wereobtained.

EXAMPLE 2 Identification of Different MRP-1 Gene Alleles by SequenceDetermination in Various Individuals

For sequence analysis of relevant regions of the human MRP-1 gene from24 different individuals, PCR amplification of the relevant fragments ofthis gene was carried out (see Table 1) and the purified PCR productssubsequently sequenced with established methods (ABI dyeterminator cyclesequencing). A very important parameter that was needed to considerusing this approach was that each normal human individual harbors twocopies of this gene. Because of this diploidy (of autosomal genes; theMRP-1 gene is an autosomal gene on chromosome 16), great care had to betaken in the evaluation of the sequences to be able to identifyunambiguously not only homozygous sequence variations but alsoheterozygous variations.

For the initial evaluation of gene variations in the human population,sequence analyses of the relevant regions of the MRP-1 gene were carriedout from the genomic DNA from 24 different individuals. This number ofindividual samples was then extended for a screening for all the MRP-1gene fragments, in which SNP's could be identified. The sequences wereinspected for the occurence of DNA sequences that were deviant from thepublished sequences of the MRP-1 gene. These reference sequences areconsidered as “wildtype” sequences in all of this work. Becausepopulation genetics enables a calculation of the expected frequency ofhomozygous vs. heterozygous alleles of a defined gene (Hardy-Weinbergdistribution, using the formulas p=(2×AA+1×Aa)/2N and p+q=1: AA=numberof probands homozygous for the wt-allele, Aa=number of heterozygotes,N=size of the sample test, p=frequency of the wt-allele, q=frequency ofthe mut-allele, q²=frequency of the genotype homozygous for themut-allele), it was possible to confirm the predicted (using theseformulas) distribution of homozygous vs. heterozygous alleles anddeviations with the experimental findings (see Table 2). This serves asinternal control and confirmation that a detected sequence deviationindeed represents a novel allele.

In total 42 new and still unpublished polymorphisms could be found inthe MRP-1 gene. The localisation of these novel SNP's in the MRP-1 geneand in the MRP-1 protein, respectively, is shown in FIG. 1. 6 of allthese new polymorphisms could be identified only in renal cell carcinoma(RCC) samples (see also example 4). The following table gives anoverview over all different types of novel MRP-1 polymorphisms, whichhave been identified in the initial screen (24 control samples, example2), as well as in the extended screen, that includes clinical samples(70 RCC samples, see example 4):

Total number of SNP location newly found SNP's comments Promoter: 11 2SNP's in RCC samples only Introns: 20 2 SNP's in RCC samples onlyExons: total 10 silent 7 1 SNP in RCC samples only amino acid 3 R723Q(splicing variant substitution region, first ATP binding domain) R433S(cytoplasmic domain) F329C (transmembrane domain no. 6; in RCC samplesonly) 3′-UTR 1

In regard to the 42 newly found SNP's, the different types ofpolymorphisms that were detected, as well as their distribution over theMRP-1 gene and the possible meaning of the new SNP's are described inmore detail below. The exact positions and further details of the novelalleles, including the exact novel sequence and sequence deviation, andthe homozygous vs. heterozygous distribution of the respective allele inthe population are listed in Table 2. The expected frequency forhomozygotes of the variant allele were calculated on the basis of theHardy-Weinberg distribution (formulas see above). The deviant base inthe sequence is bold and underlined.

The polymorphisms newly found in the MRP-1 gene might have an effecteither on the function of the MRP-1 polypeptide or its expression ortranslation. The promoter polymorphisms may especially affect thetranscription level, while the SNP which was identified in the 3′-UTRmight have an effect on the stability of the respective mRNA. Becausethe amino acid substitutions F329C, R433S and R723Q are localized inspecific functional domains of the MRP-1 polypeptide (see above in thetable), an effect of these SNP's on folding, activity or substratespecificity of the respective domains is conceivable. The singlenucleotide polymorphisms resulting in silent mutations may effectinteraction with a tRNA during translation of mRNA encoded by a genecomprising said single nucleotide polymorphisms. The polymorphisms,which could be found in the introns of the MRP-1 gene might have aneffect on splicing of MRP-1 transcripts containing said singlenucleotide polymorphisms.

The described single nucleotide polymorphisms are useful as e.g.diagnostic markers since they could be correlated with phenotypesresulting thereof, such as cancer, like kidney cancer. Furthermore thesingle nucleotide polymorphisms in MRP-1 may cause unsufficient and/oraltered drug uptake, transport or elimination.

EXAMPLE 3 Methods for Specific Detection and Diagnosis of MRP-1 Alleles

Methods to detect the various MRP-1 alleles that have been identifiedutilize the principle that specific sequence differences can betranslated into reagents for allele differentiation. These reagentsprovide the necessary backbone for the development of diagnostic tests.Examples for such reagents include—but are not limitedto—oligonucleotides that deviate from the wildtype MRP-1 sequence in thenewly identified base substitution. Frequently, the principles ofdiagnostic tests for the determination of the individual MRP-1 genestatus include—but are not limited to—differences in the hybridizationefficiencies of such reagents to the various MRP-1 alleles. In addition,differences in efficacy of such reagents in, or as different substratesfor, enzymatic reactions, e.g. ligases or polymerases or restrictionenzymes can be applied. The principles of these are well known toexperts of the field. Examples are PCR- and LCR techniques,Chip-hybridizations or MALDI-TOF analyses. Such techniques are describedin the prior art, e.g., PCR technique: Newton, (1994) PCR, BIOSScientific Publishers, Oxford; LCR-technique: Shimer, Ligase chainreaction. Methods Mol. Biol. 46 (1995), 269-278; Chip hybridization:Ramsay, DNA chips: State-of-the art. Nature Biotechnology 16 (1998),40-44; and MALDI-TOF analysis: Ross, High level multiplex genotyping byMALDI-TOF mass spectrometry, Nature Biotechnology 16 (1998), 1347-1351.Other test principles are based on the application of reagents thatspecifically recognize the MRP-1 variant as translated expressedprotein. Examples are allele-specific antibodies, peptides, substrateanalogs, inhibitors, or other substances which bind to (and in someinstances may also modify the action of) the various MRP-1 protein formsthat are encoded by the new MRP-1 alleles. The examples that arepresented here, to demonstrate the principles of diagnostic tests withreagents derived from the novel nucleotide substitutions defined in thisapplication, are based on PCR-methods. It is obvious that, applying thedescribed specific reagents, any of the other methods will also work forthe differentiation of MRP-1 alleles.

EXAMPLE 4 Distribution of MRP-1 Single Nucleotide Polymorphisms inKidney Cancer Samples

To identify potential direct correlations of MRP-1 polymorphisms withclinical relevant phenotypes in humans, totally 70 renal cell carcinoma(RCC) samples were subjected to the determination of MRP-1 polymorphismsas described in example 2. Kidney cancer is the third most frequenturological tumor, accounting in the United States for 28,000 cases inthe year 1995 and approximately 11,000 deaths each year in the US (Wingoet al. 1995, CA Cancer J Clin 45 (1): 8-30). One of the major riskfactors for sporadic RCC are somatic mutations in the VHL tumorsuppressor gene (Levine 1996, Radiol Clin North Am 34: 947-964; Linehanet al. 1995, JAMA 273: 564-570). The incidence of kidney cancerincreases continuously by 2 to 4% per year in the United States andother industrialized countries (Chow et al. 1999, JAMA 281 (17):1628-1631). These data support, that environmental factors, i.e.exposure to carcinogens, diuretic and antihypertensive drugs, tobaccosmoke and dietary constituents may be involved in the occurence of RCC(Schlehofer et al. 1996, Int. J. Cancer 66: 723-726; Heath et al. 1997,Am. J. Epidemiol 145 (7): 607-613).

As excretory organs the kidneys are committed to the detoxification andexcretion of carcinogens and metabolites. It is feasible to assume thatfactors or genes that play a role in the defense of kidney cells againstdietary and environmental toxins or metabolites may influence theindividual susceptibility towards RCC. Consequently, geneticpolymorphisms in xenobiotic-metabolizing enzymes have been reported tomodify RCC risk in the caucasian population (Longuemaux et al. 1999,Cancer Res. 59: 2903-2908). Due to its role in detoxification, the genefor the human multidrug resistance-associated protein (MRP-1) may beanother interesting candidate.

For the evaluation, if some of the newly found MRP-1 single nucleotidepolymorphisms are overrepresented and underrepresented in these kidneycancer samples, respectively, the allele distribution was determined.The allele, as well as the genotype frequencies for all new MRP-1polymorphisms distributed on the kidney cancer samples and in comparisonto that distributed on control samples are listed in the followingtable.

Frequency in % Homozygotes Frequency mutant in % (expected Sample Wt-Mut- Homozygotes Hardy SNP collection allele allele heterozygotes mutantWeinberg) T124667C Controls 62.5 37.5 50 12.5 14.1 (intron 1) RCC G1884AControls 93.3 6.7 13.3 0 0.5 (Prom1/exon RCC 1) 1720-1723delGGTAControls 87.5 12.5 25 0 1.5 (Prom 2) RCC 83.6 16.4 23.4 4.7 2.7 C1163TControls 91.3 8.7 17.4 0 0.7 (Prom 3) RCC 84.5 15.5 27.7 1.7 2.4 926insTControls 82.4 17.6 11.8 11.8 3.1 (Prom 3) RCC 62.9 37.1 51.6 11.3 13.8437insTCCTTCC Controls 97.6 2.4 4.8 0 0.1 (Prom RCC 96.3 3.7 7.4 0 0.14) A381G Controls 72.7 27.3 36.4 9.1 7.4 (Prom 5) RCC 61.5 38.5 50.813.1 14.8 G233A Controls 84.8 15.2 30.4 0 2.3 (Prom 5) RCC 77.9 22.134.4 4.9 4.9 C189A Controls 95.7 4.3 8.7 0 0.2 (Prom 5) RCC G39508AControls 93.5 6.5 13.04 0 0.4 (intron 2) RCC 92.7 7.3 11.3 1.6 0.5 C174TControls 95.8 4.2 8.3 0 0.2 (intron 6) RCC 100 0 0 0 0 C248A Controls79.2 20.8 25 8.3 4.3 (intron 7) RCC 80 20 40 0 4 C258G Controls 70.829.2 33.3 12.5 8.5 (intron 7) RCC 71.8 28.2 45.5 5.5 7.9 G79A (exonControls 93.7 6.3 12.5 0 0.4 8, Pro to RCC 96.3 3.7 7.5 0 0.1 Pro) T88C(exon Controls 72.9 27.1 37.5 8.3 7.3 8, Val to Val) RCC 71.3 28.7 42.67.4 8.2 T249G (exon RCC 99.3 0.7 1.5 0 0.01 8, (only in Phe329Cys) thesesamples) *T95C (exon Controls 71.7 28.3 39.1 8.7 7.9 9, Asn to RCC 73.126.9 44.8 4.5 7.2 Asn) *A259G Controls 71.7 28.3 39.1 8.7 7.9 (intron 9)RCC 73.9 26.1 43.3 4.5 6.8 G57998T Controls 96.9 3.1 6.3 0 0.1 (exon 10,RCC 99.3 0.7 1.5 0 0.01 Arg433Ser) C57853T Controls 97.9 2.1 4.2 0 0.1(intron 10) RCC 97.1 2.9 5.8 0 0.1 C53282G Controls 77.1 22.9 37.5 4.25.3 (intron 11) RCC 73.8 26.2 46.2 3.1 6.8 *A137710G Controls 79.2 20.833.3 4.2 4.4 (intron 12) RCC 81.5 18.5 29.6 3.7 3.4 *C137667T Controls79.2 20.8 33.3 4.2 4.4 (exon 13, RCC 81.5 18.5 29.6 3.7 3.4 Leu to Leu)C137647T Controls 85.4 14.6 29.2 0 2.1 (exon 13, Tyr RCC 94.4 5.6 7.41.9 0.3 to Tyr) *G27258A Controls 95.8 4.2 8.3 0 0.2 (exon 17, RCC 96.23.8 7.7 0 0.2 Arg723Gln) *34207delAT Controls 95.8 4.2 8.3 0 0.2 (intron18) RCC 96.3 3.7 7.4 0 0.1 G34215C Controls 84.8 15.2 30.4 0 2.3 (intron18) RCC 84.3 15.7 25.7 2.9 2.5 55156insTGGGC Controls 75 25 0 25 6.3(intron RCC 77.6 22.4 0 22.4 5.03 21) T55472C Controls 83.3 16.7 8.312.5 2.8 (intron 22) RCC 78.6 21.4 10.7 16.1 4.6 G14008A Controls 80.419.6 39.1 0 3.8 (exon 28, RCC 73.3 26.7 44.2 4.7 7.2 Ser to Ser)G150727A Controls 66.7 33.3 50 8.3 10.9 (intron 28) RCC 55 45 44.3 22.920.3 17970delT Controls 75 25 41.7 4.2 6.3 (intron 29) RCC 75.7 24.334.3 7.2 5.9 G18195A Controls 73.3 26.7 40 6.7 7.1 (intron 30) RCC 80.419.6 21.7 8.7 3.8 G21133A (3′ Controls 97.9 2.1 4.2 0 0.1 flanking RCC95.7 4.3 8.7 0 0.2 region) G38646C Controls 73.3 26.7 53.3 0 7.1(Prom 1) RCC G34218A RCC 96.3 3.7 7.4 0 0.1 (intron 18) (only in thesesamples) C18067T RCC 98.9 1.1 2.2 0 0.02 (exon 30, Ala (only in to Ala)these samples) C440T RCC 99.3 0.7 1.5 0 0.01 (Prom 5) (only in thesesamples) C1625A RCC 96.9 3.1 6.3 0 0.1 (prom 2) (only in these samples)C17900T RCC 97.9 2.1 4.3 0 0.1 (intron 29) (only in these samples)

Three pairs of linked polymorphisms are listed in this table, whereaseach SNP is marked by an asterics. In regard to their under- andoverrepresentation in the RCC samples in comparison to the controlsamples, respectively, all of the new single nucleotide polymorphismsare of great interest, because they represent genetic variety in humans,which may serve as potential targets for diagnosis and therapy and asrisk factors for kidney cancer. Some examples: in contrast to thecontrol samples the mutant alleles of 4 promoter SNP's found in theMRP-1 gene (C1163T (Prom 3), 926insT (Prom 3), A381G (Prom 5) and G233A(Prom 5)) are overrepresented in the RCC sample group. Likewise some ofthe new intron SNP's, like G150727A (intron 28) and T55472C (intron 22),as well as the silent mutation G14008A (exon 28, Ser to Ser) show alleledistributions, which point to correlation with kidney cancer. Inaddition, especially the 6 SNP's, which could be only detected in theRCC samples may have an impact for the diagnosis and therapy of kidneycancer.

EXAMPLE 5 Statistical Analyses of Correlations Between MRP-1 SingleNucleotide Polymorphisms and Renal Cell Carcinoma (RCC)

Statistical evaluations were performed in regard to the presence ofSNP's in RCC samples compared to their frequencies in a controlpopulation. For this purpose, 70 RCC samples and 24 control samples werecompared. Statistical analysis was performed using the computer programSPSS 10.0 (SPSS, Chicago, USA). This evaluation results in statisticallysignificant correlations of definite SNP's with the existence of renalcell carcinoma (RCC).

The p-values of the statistical evaluation (Chi-Quadrat-Test), whichresult in genotype/phenotype correlations are:

gene SNP Controls vs. RCC, p-value MRP-1 926insT (Promoter) 0.005 G79A(exon 8) 0.063 C137647T (exon 0.039 13)

EXAMPLE 6 Effects of Kidney Cancer Associated MRP-1 Polymorphisms onDrug Transport Activity and Pharmacology

As excretory organs the kidneys are committed to the detoxification andexcretion of watersoluble carcinogens and metabolites. Therefore,factors or genes that influence the individual susceptibility towardskidney cancer are related to the defense capacity of kidney cellsagainst dietary and environmental toxins or metabolites (Epidauros MDR-1risk factor patent). Among these factors, the gene for theP-glycoprotein (Pgp), which transports toxic substances, has been shownto confer a significant risk factor for kidney cancer, such as for RCC,if it is present in an allelic version that corresponds to low transportactivity (Epidauros MDR-1 risk factor patent).

The multidrug resistance-associated protein 1 (MRP-1) is, like MDR-1expressed in the renal tubular cells of the kidney and extrude differentclasses of substances in an ATP dependent manner from the inside to theoutside of plasma membranes within these cells. The physiological roleof this energy-dependent export mechanism in the kidney is theprotection of cells. The fact that, like MDR-1 SNP's, also polymorphismsin the MRP-1 gene (which has a very similar function) confersignificantly increased risk to develop kidney cancer, such as RCC (seetables in examples 2, 4 and the results of the statistical evaluation inexample 5, respectively), indicates the underlying molecular mechanismto be the same for the functional polymorphisms in MDR-1 as well as inMRP-1: altered and/or reduced transport capacities lead to increasedexposure of renal cells to carcinogenic, toxic and/or noxic substances,which is responsible for the increased risk to develop malignant changesin tubular cells. Beside the Promoter SNP 926insT, which shows astatistically significant correlation with RCC (p=0.005), also thefollowing MRP-1 promoter SNP's C1163T, A381G and G233A, which areoverrepresented in RCC are good candidates for such risk factors.

Variable transport capacities of MRP-1-variants play a role not only ininfluencing the individual risk of developing kidney cancer, such asRCC, but such variations will also affect individual pharmacologicalresponses to medications. For example, the expression of MRP-1correlates with therapy outcome in cancer therapy: Higher MRP-1 activityleads to a resistance of the cell against MRP-1 substrates. Thismultidrug resistance could be shown for numerous MRP-1 substrates.

Therefore, MRP-1 polymorphisms, especially those with functionalimportance, even up to a degree that associated with increased risk forkidney cancer, such as RCC due to a decreased capacity of tubular cellsto clear damaging agents, are important for predicting clearance anduptake of MRP-1 substrates, or drugs whose metabolites are MRP-1substrates (see FIGS. 2 A and B).

EXAMPLE 7 Correlation of MRP-1 Polymorphisms with MRP-1 Expression andSide Effects During Therapy with MRP-1 Substrates

Functional polymorphisms in the MRP-1 gene (see tables in examples 2 and4) affect the transport activity and subsequently the levels of drugswhich are substrates of MRP-1. Increased levels of such drugs can leadto side effects whereas decreased levels may result in subtherapeuticaldrug levels that lead to therapy failure. Three different patientcollectives, two show side effects during drug therapy and one for whichthe MRP-1 mRNA levels had been defined, were analyzed to determinewhether MRP-1 polymorphisms correlate with transporter activity andsubsequently with alterations in drug activities and side effects.Statistical evaluations were performed in regard to the presence ofSNP's in these collectives with side effects during drug therapy andincreased/decreased mRNA levels compared to their frequencies in controlsamples. For this purpose, the 3 collectives (collective 1: samples withnephrotoxicities after cisplatin therapy; collective 2: liver and kidneyside effects; collective 3: samples with defined high or low MRP-1 mRNAlevels) were screened for all MRP-1 gene fragments, in which the newSNP's could be detected. For those of the newly identified MRP-1 SNP'swhich are overrepresented or underrepresented, the allele distributionwas determined. As an example, the allele and genotype frequencies forone MRP-1 polymorphism are listed in the following table for collective2 and compared to control samples:

Frequency in % Homozygotes Frequency mutant in % (expected Sample Wt-Mut- Homozygotes Hardy SNP collection allele allele heterozygotes mutantWeinberg) G150727A Controls 66.7 33.3 50 8.3 10.9 (intron 28) Collective2 50 50 14.3 42.9 25

In contrast to control samples the mutant allele (150727A) of one SNPfound in the MRP-1 gene (G150727A, intron 28) is overrepresented in thesamples of collective 2. Statistical evaluations were performed inregard to the presence of this SNP in samples with liver and kidney sideeffects (collective 2) compared to their frequencies in a controlpopulation. The statistical analysis was performed using the computerprogram SPSS 10.0 (SPSS, Chicago, USA). This evaluation results in astatistically significant correlation of a definite SNP with liver andkidney side effects.

The genotyp/phenotype correlation is confirmed by the p-value of thestatistical evaluation (Chi-Quadrat-Test):

Controls vs. liver and kidney gene SNP side effects, p-value MRP-1G150727A 0.044 (intron 28)

Furthermore, a correlation of MRP-1 gene variants and mRNA expression ofMRP-1 could be found for two new MRP-1 SNP's (T95C, exon 9, Asn to Asnand A259G, intron 9). These are linked SNP's (see also table in example4). As shown in FIG. 3 (Diagram A and B), the mutant allele correlateswith decreased MRP-1 mRNA expression. Thus, the analysis of thesefunctional important SNP's is of high diagnostic/prognostic value,because it allows the prediction of therapy outcome and side effects,and of expression levels of MRP-1.

EXAMPLE 8 MRP1 Genotypes in Patients Suffering from Drug-Induced HepaticToxicity

MRP1 genotypes were investigated in patients suffering from drug-inducedhepatic toxicity (n=7) and healthy controls (n=95). Pearson chi-squarewas calculated from contingency tables to test the equality ofproportions between patients and controls. When appropiate Fisher'sExact Test was applied. The level of significance was set to p=0.05.Statistical analysis was performed using SPSS 10.1 (SPSS, Chicago, USA).The level of significance was set to p=0.05.

Three SNPs (T>C₉₅, A>G₂₅₉, and C>G₅₃₂₈₂) were found to be associatedwith the occurrence of liver toxicity. The frequency of homozyguoslymutant genotypes was statistically significant elevated as summarized inthe following table.

Frequency Distribution of MRP1 Genotypes SNP Controls [%] Controls [%]wt > mut_(position) AccNo¹ SeqID N wt/wt wt/m m/m N wt/wt wt/m m/m P²T > C₉₅ AF022831 171 7 47.8 44.6 7.6 92 85.7 14.3 0.035 A > G₂₅₉AF022831 177 7 47.8 44.6 7.6 92 85.7 14.3 0.035 C > G₅₃₂₈₂ GI: 7209451195 6 55.3 40.4 4.3 94 85.3 16.7 0.05 ¹Accession Number of referencesequence (wt allele) ²P value of statistical test wt/wt homozygouswildtypes wt/m heterozygots m/m homozygous mutants

Two of these SNPs are linked (T>C₉₅ and A>G₂₅₉) and have beendemonstrated (example 7) to correlate with decreased MRP1 expression. Itcan be concluded that a reduced hepatic MRP1 expression leads to adecreased capacity of hepatocytes to transport toxic substrates with theconsequence of an elevated risk to hepatocellular damage. Thus, SNPs inthe MRP1 can explain interindividual variations in the susceptibility toadverse drug events (ADEs) and are important diagnostic markers topredict the individual risk of patients in order to prevent patientsfrom ADEs by e.g. dosage adjustments or switching to other medications.

EXAMPLE 9 MRP1 Genotypes in Patients Suffering from Renal Carcinoma(RCC)

MRP1 genotypes were investigated in patients suffering from renalcarcinoma (RCC) and healthy controls. Pearson chi-square was calculatedfrom contingency tables to test the equality of proportions between RCCand controls. When appropiate Fisher's Exact Test was applied. The levelof significance was set to p=0.05. Statistical analysis was performedusing SPSS 10.1 (SPSS, Chicago, USA). Pearson chi-square was calculatedto test the equality of proportions. The level of significance was setto p=0.05.

Three SNPs have been already described to be correlated with RCC inexample 5. Additionally, the nucleotide substitution A>G₃₈₁ was found tobe statistically significant associated with RCC and T>C₁₂₄₆₆₇ tended tobe associated with renal carcinoma confirming further the important roleof MRP1 for pharmacology and toxicology of drugs.

Frequency Distribution of MRP1 Genotypes SNP Patients [%] Controls [%]wt > mut_(position) AccNo¹ SeqID N wt/wt wt/m m/m N wt/wt wt/m m/m P²T > C₁₂₄₆₆₇ AC026452 075 33 45.5 51.5 3.0 90 57.8 31.1 11.1 0.075 A >G₃₈₁ U07050 111 59 35.6 52.5 11.9 88 53.6 32.1 14.3 0.027 ¹AccessionNumber of reference sequence (wt allele) ²P value of statistical testwt/wt homozygous wildtypes wt/m heterozygots m/m homozygous mutants

TABLE 1 Primers for the amplification of fragments of the MRP1 genePrimer sequence PCR fragment (5′ to 3′ PCR Fragment namePCR primer position orientation) condition size Accession numberAC026452 Exon1/Prom 1 38590-38608 MRP1-P1f GTA GGG GGC TCC GTT CAC G B880 bp 124576-124600 MRP1-E1r2 CCT GGA AGG TTG TTT TTA CAG ACG GAccession number U07050 Promoter fragment 1359-1377 MRP1-P2fTGG AGA CTG GCG CCG TCT G C 408 bp 2 1767-1746 MRP1-P2rAAG GAC AGT ATC CGT CAC CAG G Promoter fragment 830-851 MRP1-P3fCAT GGG GTT GTG AGG ATT GCA C A 590 bp 3 1423-1401 MRP1-P3rTGA GAT TCA AAC CCG TGA GCA GC Promoter fragment 351-374 MRP1-P4fCTT AGA AAC TCA TTC ACC CTT A 550 bp 4 GGG 902-881 MRP1-P4rGTG ACA AGG CTT CCT AAG GCT G Promoter fragment 144-170 MRP1-P5fGAT TAA CAT CTG CCA TCT TAC CAT A 321 bp 5 AAG 465-445 MRP1-P5rCCT CCC CCC AAT CAA AGG ACC GI number 7209451 Exon 2 39769-39789MRP1-E2f1 AGC TGG TTT CAT GCT CCA GGC A 374 bp 39416-39440 MRP1-E2r1CTA GAA GAA GGA ACT TAG GGT CAA C Accession number AF022825 Exon 3 24-44MRP1-E3f TTC CAG GGC GGT CTG TTG TAG A 233 bp 257-235 MRP1-E3rATT ACT TTT GGT CTC CAC TGA GC Accession number AF022826 Exon 4 68-90MRP1-E4f2 AAA ACC CAA CAA CTC CTG TCT TG A 230 bp 297-278 MRP1-E4rGCA TCT TTC CCT CCG GGT CC Accession number AF022827 Exon 5 35-55MRP1-E5f1 ACC CAG CCC CAG AAT GTG ATC A 206 bp 240-219 MRP1-E5r2GCA CAC ACA CTC ATT TGT GGT C Accession number AF022828 Exon 6  4-26MRP1-E6f GAG CAG CTG ACT ACT TGC TAA GC A 209 bp 212-190 MRP1-E6r1CAT TCA TTC ATT CAC TCC CCA CC Accession number AF022829 Exon 7 17-41MRP1-E7f CTG TCA TTG ACT CTC ATT GCC TAA A 279 bp C 295-275 MRP1-E7r1AGT AAC AGG CAG CAC TGC CAG Accession number AF022830 Exon 8 29-49MRP1-E8f ATC TCT GGC AGA CCC CAC AAC A 336 bp 364-341 MRP1-E8r1AAC TGA AAG ATC AAA GCC AAG GAG Accession number AF022831 Exon 9 26-47MRP1-E9f CCC CAC GTG TCA CAA GTC ATT C A 322 bp 347-328 MRP1-E9rTGG GCT GGA AAT CCC CAC GC GI number 7209451 Exon 10 58203-58184MRP1-E10f1 GGG AGG AGG AGA GAT CTG CG A 413 bp 57791-57810 MRP1-E10r1TGA ACC ACA GCC GGA ACT GC GI number 7209451 Exon 11 53578-53559MRP1-E11f GGA TGG ATC AAC CGG GGA AG A 353 bp 53226-53248 MRP1-E11rTCA GAA TCC CAG ATA TGC AGC CG GI number 7209451 Exon 12 22183-22204MRP1-E12f1 TGT TGA GTG ATG GGC TGA TCC C A 344 bp 22526-22499 MRP1-E12rCCT TTT AAA AAT ATT CAG GTA CGC AGA G Accession number AC003026 Exon 1311927-11949 MRP1-E13f CAC TGC TCC TAG GAT GAT GAC TC A 312 bp12238-12218 MRP1-E13r GAG TGT GAT CTA GAG GCT GCG Exon 14 15397-15419MRP1-E14f GGG GAA ACC CTT GAA AGT TAA CC A 264 bp 15660-15638 MRP1-E14rCAG CCA AGG GAA AGA AAT GCA AG Exon 15 20044-20063 MRP1-E15fATG CCT AGC GCC ATT CGT GC A 285 bp 20328-20309 MRP1-E15rGGG AGC ACG GTG GGA ATT CG Exon 16 23040-23063 MRP1-E16fGAA GGA ATG TTG AGG CCT TCA A 402 bp GTG 23441-23418 MRP1-E16rGAA AAG AGA CGT TGC TGC TTT CGC Exon 17 27108-27128 MRP1-E17fAAG TGA GGC CCT CCT AGC AGG C 372 bp 27479-27458 MRP1-E17rTGA TAG CAG CAG ACT CAC AGC C Exon 18 30588-30607 MRP1-E18fACA CTC GGC CTG CTT CTA CG A 326 bp 30913-30892 MRP1-E18rAAG GAC TCC TAA AGG GGA CAC G Exon 19 34085-34105 MRP1-E19fGCT CCT GGA TGC TGT TAT CGC A 430 bp 34514-34495 MRP1-E19r2TGG CTG GTG GCA ACC TCA AAG Accession number AC003026 Exon 2046405-46427 MR-E20f2 CCC TTG GTT TTA GCA TCT GCC TC A 239 bp 46643-46621MR-E20r GGG CTG AGG CCT TTT TTT GTT CC Exon 21 50449-50471 MRP1-E21fTGT GTG CAT GTG GAA ACA CTC CG A 368 bp 50816-50792 MRP1-E21rGAC AGG TGA GTT AAC ATA GAC AAG G Accession number AC003026 Exon 2255116-55134 MRP1-E22f TGC TGG TGA AGC CCC CGA C A 402 bp 55517-55497MRP1-E22r GTT TGG GGT CCC ACA AAA CGC Exon 23 58530-58548 MRP1-E23f3CTC CCT GCA GTG CCT GGT C A 474 bp 59003-58983 MRP1-E23r3CCA CAC TGG GGA CAT GGT AAG Exon 24 65670-65688 MRP1-E24f1AGG GCA GCC CGG CTC TAA C A 444 bp 66113-66093 MRP1-E24rGCC GGG GTT TGG CTT TAT ACC Accession number U91318 Exon 25 4270-4292MRP1-E25f CTC TCT CTG GAA TTA CTG CGG AG A 385 bp 4654-4634 MRP1-E25rCTG CTC CTC AAA CTC CGT ACC Exon 26 5371-5393 MRP1-E26fGAA AGT CAA GTA CGC CCG CTT AC A 242 bp 5612-5593 MRP1-E26rAGG TGC ACA GGA TAG GGT CC Exon 27 11200-11220 MRP1-E27fCTG AGA GGG TGC TCT GTA TCG A 545 bp 11744-11721 MRP1-E27rCAC TTC TGC AAG TTG TAT GCG CTC Exon 28 13844-13863 MR-E28fGAG AGG GCT GTC GAG TTG GG C 349 bp 14192-14170 MR-E28rTCA GTG CAA TCA TAG GGC TTG CC Accession number U91318 Exon 2916017-16036 MR-E29f CCA GAA GTC CTT AGG TCG CC A 317 bp 16333-16311MR-E29r CTT CAA ACA CCC CTA CCG AGA TG Exon 30 17859-17880 MR-E30fGGA CAT GCT TTC CTG GTC AAG C A 430 bp 18288-18268 MR-E30rGGG CTG TCA CTA GGG ATA AGG Exon 31, (incl. 3′- 20650-20670 MR-E31fGCA ACC AGC TGG AAG GTA CTG A 592 bp UTR) 21241-21219 MR-E31rCAG AAG TCT GGC TGC CAA AAC TC

Conditions for the Different PCR Fragments:

PCRs were carried out for all fragments in a reaction volume of 50 μl.40 ng DNA template was added to standard PCR buffer containing 1.5 mMMgCl2 (Qiagen, Hilden), 200 μM dNTP's (Roth, Karlsruhe), 0.4 μM(conditions A and C) or 1.6 μM (condition B) of each primer (Metabion,Munich), 10 μl Q-Solution (condition C; Qiagen, Hilden), 4 μl DMSO(condition B) and 1 U Taq polymerase (Qiagen, Hilden). All PCRs(conditions A and C) were performed on a Perkin Elmer thermocycler(model 9700) with an initial denaturation step of 2 min at 94° C. and 34amplification cycles of denaturation at 94° C. for 45 sec, primerannealing at 62° C. for 45 sec, and 1 min for 72° C. followed by a finalextension of 72° C. for 10 min. In the case of condition B the PCRreaction was performed with an initial denaturation step of 3 min at 96°C. and 35 amplification cycles of denaturation at 96° C. for 45 sec,primer annealing at 62° C. for 30 sec, and 1 min for 72° C. followed bya final extension of 72° C. for 10 min.

TABLE 2 New SNP's in the gene for MRP1 Position of PCR fragment the namevariation wt-sequence wt/mut- and/or mut-sequence Accession numberAC026452 wt/mut: Exon 1/Prom 1 124667 f: GCGTGCCCAG T CCTGGGGTTTf: GCGTGCCCAG T/C CCTGGGGTTT (intron 1) (SNP 34) (SEQ ID No: 071)(SEQ ID No: 073) r: AAACCCCAGG A CTGGGCACGC r: AAACCCCAGG A/G CTGGGCACGC(SEQ ID No: 072) (SEQ ID No: 074) mut/mut: f: GCGTGCCCAG C CCTGGGGTTT(SEQ ID No: 075) r: AAACCCCAGG G CTGGGCACGC (SEQ ID No: 076) Accessionnumber U07050 wt/mut: Exon 1/Prom 1 1884 (SNP f: AGCCTTGGAG G ATCTGGGGTGf: AGCCTTGGAG G/A ATCTGGGGTG 33) (SEQ ID No: 077) (SEQ ID No: 079)r: CACCCCAGAT C CTCCAAGGCT r: CACCCCAGAT C/T CTCCAAGGCT (SEQ ID No: 078)(SEQ ID No: 080) mut/mut: f: AGCCTTGGAG A ATCTGGGGTG (SEQ ID No: 081)r: CACCCCAGAT T CTCCAAGGCT (SEQ ID No: 082) wt/mut: Promoter 1720-1723f: ACTCCAGGCA GGTA GGGGGCTCCG f: ACTCCAGGCA GGTA/delGGTA GGGGGCTCCGfragment 2 del GGTA (SEQ ID No: 083) (SEQ ID No: 085) (SNP 25)r: CGGAGCCCCC TACC TGCCTGGAGT r: CGGAGCCCCC TACC/delTACC TGCCTGGAGT(SEQ ID No: 084) (SEQ ID No: 086) mut/mut: f: ACTCCAGGCA delGGTAGGGGGCTCCG (SEQ ID No: 087) r: CGGAGCCCCC delTACC TGCCTGGAGT(SEQ ID No: 088) Accession number U07050 wt/mut: Promoter 1163f: TGTGATCGGC C CGCCTCGGCT f: TGTGATCGGC C/T CGCCTCGGCT fragment 3(SNP22) (SEQ ID No: 089) (SEQ ID No: 091) r: AGCCGAGGCG G GCCGATCACAr: AGCCGAGGCG G/A GCCGATCACA (SEQ ID No: 090) (SEQ ID No: 092) mut/mut:f: TGTGATCGGC T CGCCTCGGCT (SEQ ID No: 093) r: AGCCGAGGCG A GCCGATCACA(SEQ ID No: 094) wt/mut: Promoter 926 (SNP f: TTAATTTTTT T ATTATTATTTf: TTAATTTTTT T/insT ATTATTATTT fragment 3 21) (SEQ ID No: 095)(SEQ ID No: 097) r: AAATAATAAT A AAAAAATTAA r: AAATAATAAT A/insAAAAAAATTAA (SEQ ID No: 096) (SEQ ID No: 098) mut/mut: f: TTAATTTTTTTinsT ATTATTATTT (SEQ ID No: 099) r: AAATAATAAT insA AAAAAAATTAA(SEQ ID No: 100) wt/mut: Promoter 437 (SNP f: TTCCTCCTTC C CTCGCTAGGTf: TTCCTCCTTC C/insTCCTTCC CTCGCTAGGT fragment 4 31) (SEQ ID No: 101)(SEQ ID No: 103) r: ACCTAGCGAG G GAAGGAGGAA r: ACCTAGCGAG G/insAGGAAGGGAAGGAGGAA (SEQ ID No: 102) (SEQ ID No: 104) mut/mut: f: TTCCTCCTTCCTCCTTCC CTCGCTAGGT (SEQ ID No: 105) r: ACCTAGCGAG GGAAGGA GGAAGGAGGAA(SEQ ID No: 106) Accession number U07050 wt/mut: Promoter 381 (SNPf: TGGGGGACCC A GGCCAATAAA f: TGGGGGACCC A/G GGCCAATAAA fragment 520/30) (SEQ ID No: 107) (SEQ ID No: 109) r: TTTATTGGCC T GGGTCCCCCAr: TTTATTGGCC T/C GGGTCCCCCA (SEQ ID No: 108) (SEQ ID No: 110) mut/mut:f: TGGGGGACCC G GGCCAATAAA (SEQ ID No: 111) r: TTTATTGGCC C GGGTCCCCCA(SEQ ID No: 112) wt/mut: Promoter 233 (SNP f: AAGAGTAGCA G TTTTATCTTGf: AAGAGTAGCA G/A TTTTATCTTG fragment 5 19) (SEQ ID No: 113)(SEQ ID No: 115) r: CAAGATAAAA C TGCTACTCTT r: CAAGATAAAA C/T TGCTACTCTT(SEQ ID No: 114) (SEQ ID No: 116) mut/mut: f: AAGAGTAGCA A TTTTATCTTG(SEQ ID No: 117) r: CAAGATAAAA T TGCTACTCTT (SEQ ID No: 118) wt/mut:Promoter 189 (SNP f: AAAAAAATCC C AATCCAAAAA f: AAAAAAATCC C/AAATCCAAAAA fragment 5 35) (SEQ ID No: 119) (SEQ ID No: 121)r: TTTTTGGATT G GGATTTTTTT r: TTTTTGGATT G/T GGATTTTTTT (SEQ ID No: 120)(SEQ ID No: 122) mut/mut: f: AAAAAAATCC A AATCCAAAAA (SEQ ID No: 123)r: TTTTTGGATT T GGATTTTTTT (SEQ ID No: 124) GI number 7209451 wt/mut:Exon 2 (intron 2) 39508 (SNP f: GTTTCGTTGT G GGGGGTGGGA f: GTTTCGTTGTG/A GGGGGTGGGA 1) (SEQ ID No: 125) (SEQ ID No: 127) r: TCCCACCCCC CACAACGAAAC r: TCCCACCCCC C/T ACAACGAAAC (SEQ ID No: 126)(SEQ ID No: 128) mut/mut: f: GTTTCGTTGT A GGGGGTGGGA (SEQ ID No: 129)r: TCCCACCCCC T ACAACGAAAC (SEQ ID No: 130) Accession number AF022828wt/mut: Exon 6 (intron 6) 174 (SNP f: CCAGGCCCCC C AGACCTCAGGf: CCAGGCCCCC C/T AGACCTCAGG 10) (SEQ ID No: 131) (SEQ ID No: 133)r: CCTGAGGTCT G GGGGGCCTGG r: CCTGAGGTCT G/A GGGGGCCTGG (SEQ ID No: 132)(SEQ ID No: 134) mut/mut: f: CCAGGCCCCC T AGACCTCAGG (SEQ ID No: 135)r: CCTGAGGTCT A GGGGGCCTGG (SEQ ID No: 136) Accession number AF022829wt/mut: Exon 7 (intron 7) 248 (SNP 2) f: CCTTTCCACT C CTGTGGCCTCf: CCTTTCCACT C/A CTGTGGCCTC (SEQ ID No: 137) (SEQ ID No: 139)r: GAGGCCACAG G AGTGGAAAGG r: GAGGCCACAG G/T AGTGGAAAGG (SEQ ID No: 138)(SEQ ID No: 140) mut/mut: f: CCTTTCCACT A CTGTGGCCTC (SEQ ID No: 141)r: GAGGCCACAG T AGTGGAAAGG (SEQ ID No: 142) wt/mut: Exon 7 (intron 7)258 (SNP 3) f: CCTGTGGCCT C AATCCAGGAT f: CCTGTGGCCT C/G AATCCAGGAT(SEQ ID No: 143) (SEQ ID No: 145) r: ATCCTGGATT G AGGCCACAGGr: ATCCTGGATT G/C AGGCCACAGG (SEQ ID No: 144) (SEQ ID No: 146) mut/mut:f: CCTGTGGCCT G AATCCAGGAT (SEQ ID No: 147) r: ATCCTGGATT C AGGCCACAGG(SEQ ID No: 148) Accession number AF022830 wt/mut: Exon 8 79 (SNP 4)f: CCAGGCAGCC G GTGAAGGTTG f: CCAGGCAGCC G/A GTGAAGGTTG (SEQ ID No: 149)(SEQ ID No: 151) r: CAACCTTCAC C GGCTGCCTGG r: CAACCTTCAC C/T GGCTGCCTGG(SEQ ID No: 150) (SEQ ID No: 152) mut/mut: f: CCAGGCAGCC A GTGAAGGTTG(SEQ ID No: 153) r: CAACCTTCAC T GGCTGCCTGG (SEQ ID No: 154) Accessionnumber AF022830 wt/mut: Exon 8 88 (SNP 5) f: CGGTGAAGGT T GTGTACTCCTf: CGGTGAAGGT T/C GTGTACTCCT (SEQ ID No: 155) (SEQ ID No: 157)r: AGGAGTACAC A ACCTTCACCG r: AGGAGTACAC A/G ACCTTCACCG (SEQ ID No: 156)(SEQ ID No: 158) mut/mut: f: CGGTGAAGGT C GTGTACTCCT (SEQ ID No: 159)r: AGGAGTACAC G ACCTTCACCG (SEQ ID No: 160) wt/mut: Exon 8 249 (SNPf: CTCATGAGCT T CTTCTTCAAG f: CTCATGAGCT T/G CTTCTTCAAG 37)(SEQ ID No: 161) (SEQ ID No: 163) (only in r: CTTGAAGAAG A AGCTCATGAGr: CTTGAAGAAG A/C AGCTCATGAG RCC (SEQ ID No: 162) (SEQ ID No: 164)samples) mut/mut: f: CTCATGAGCT G CTTCTTCAAG (SEQ ID No: 165)r: CTTGAAGAAG C AGCTCATGAG (SEQ ID No: 166) Accession number AF022831wt/mut: Exon 9 95 (SNP 6) f: AGTTCGTGAA T GACACGAAGG f: AGTTCGTGAA T/CGACACGAAGG (SEQ ID No: 167) (SEQ ID No: 169) r: CCTTCGTGTC A TTCACGAACTr: CCTTCGTGTC A/G TTCACGAACT (SEQ ID No: 168) (SEQ ID No: 170) mut/mut:f: AGTTCGTGAA C GACACGAAGG (SEQ ID No: 171) r: CCTTCGTGTC G TTCACGAACT(SEQ ID No: 172) wt/mut: Exon 9 (intron 9) 259 (SNP 7) f: AAGGTAGGGG ACGCTGTGCCA f: AAGGTAGGGG A/G CGCTGTGCCA (SEQ ID No: 173)(SEQ ID No: 175) r: TGGCACAGCG T CCCCTACCTT r: TGGCACAGCG T/C CCCCTACCTT(SEQ ID No: 174) (SEQ ID No: 176) mut/mut: f: AAGGTAGGGG G CGCTGTGCCA(SEQ ID No: 177) r: TGGCACAGCG C CCCCTACCTT (SEQ ID No: 178) GI number7209451 wt/mut: Exon 10 57998 (SNP f: ACGCTCAGAG G TTCATGGACTf: ACGCTCAGAG G/T TTCATGGACT 11) (SEQ ID No: 179) (SEQ ID No: 181)r: AGTCCATGAA C CTCTGAGCGT r: AGTCCATGAA C/A CTCTGAGCGT (SEQ ID No: 180)(SEQ ID No: 182) mut/mut: f: ACGCTCAGAG T TTCATGGACT (SEQ ID No: 183)r: AGTCCATGAA A CTCTGAGCGT (SEQ ID No: 184) wt/mut: Exon 10 (intron57853 (SNP f: GGCAGTGGGC C GAGGGAGTGG f: GGCAGTGGGC C/T GAGGGAGTGG 10)8) (SEQ ID No: 185) (SEQ ID No: 187) r: CCACTCCCTC G GCCCACTGCCr: CCACTCCCTC G/A GCCCACTGCC (SEQ ID No: 186) (SEQ ID No: 188) mut/mut:f: GGCAGTGGGC T GAGGGAGTGG (SEQ ID No: 189) r: CCACTCCCTC A GCCCACTGCC(SEQ ID No: 190) wt/mut: Exon 11 (intron 53282 (SNP f: GCCAGTTGGA CTCACTTGGGG f: GCCAGTTGGA C/G TCACTTGGGG 11) 12) (SEQ ID No: 191)(SEQ ID No: 193) r: CCCCAAGTGA G TCCAACTGGC r: CCCCAAGTGA G/C TCCAACTGGC(SEQ ID No: 192) (SEQ ID No: 194) mut/mut: f: GCCAGTTGGA G TCACTTGGGG(SEQ ID No: 195) r: CCCCAAGTGA C TCCAACTGGC (SEQ ID No: 196) Accessionnumber AC026452 wt/mut: Exon 13 (intron 137710 f: ACTCTCACTC AGGGCACAGCA f: ACTCTCACTC A/G GGGCACAGCA 12) (SNP 26) (SEQ ID No: 197)(SEQ ID No: 199) r: TGCTGTGCCC T GAGTGAGAGT r: TGCTGTGCCC T/C GAGTGAGAGT(SEQ ID No: 198) (SEQ ID No: 200) mut/mut: f: ACTCTCACTC G GGGCACAGCA(SEQ ID No: 201) r: TGCTGTGCCC C GAGTGAGAGT (SEQ ID No: 202) Accessionnumber AC026452 wt/mut: Exon 13 137667 f: GCAGGTGGCC C TGTGCACATTf: GCAGGTGGCC C/T TGTGCACATT (SNP 13) (SEQ ID No: 203) (SEQ ID No: 205)r: AATGTGCACA G GGCCACCTGC r: AATGTGCACA G/A GGCCACCTGC (SEQ ID No: 204)(SEQ ID No: 206) mut/mut: f: GCAGGTGGCC T TGTGCACATT (SEQ ID No: 207)r: AATGTGCACA A GGCCACCTGC (SEQ ID No: 208) wt/mut: Exon 13 137647f: TTGCCGTCTA C GTGACCATTG f: TTGCCGTCTA C/T GTGACCATTG (SNP 14)(SEQ ID No: 209) (SEQ ID No: 211) r: CAATGGTCAC G TAGACGGCAAr: CAATGGTCAC G/A TAGACGGCAA (SEQ ID No: 210) (SEQ ID No: 212) mut/mut:f: TTGCCGTCTA T GTGACCATTG (SEQ ID No: 213) r: CAATGGTCAC A TAGACGGCAA(SEQ ID No: 214) Accession number AC003026 wt/mut: Exon 17 (intron27159 (SNP: f: TCGTTGATCA G ATCTGTCTGT f: TCGTTGATCA G/C ATCTGTCTGT 16)mr-v-024) (SEQ ID No: 215) (SEQ ID No: 217) r: ACAGACAGAT C TGATCAACGAr: ACAGACAGAT C/G TGATCAACGA (SEQ ID No: 216) (SEQ ID No: 218) mut/mut:f: TCGTTGATCA C ATCTGTCTGT (SEQ ID No: 219) r: ACAGACAGAT G TGATCAACGA(SEQ ID No: 220) wt/mut: Exon 17 27258 (SNP f: GATTCTCTCC G AGAAAACATCf: GATTCTCTCC G/A AGAAAACATC 9) (SEQ ID No: 221) (SEQ ID No: 223)r: GATGTTTTCT C GGAGAGAATC r: GATGTTTTCT C/T GGAGAGAATC (SEQ ID No: 222)(SEQ ID No: 224) mut/mut: f: GATTCTCTCC A AGAAAACATC (SEQ ID No: 225)r: GATGTTTTCT T GGAGAGAATC (SEQ ID No: 226) Accession number AC003026wt/mut: Exon 19 (intron 34206/34207 f: AGTCTCACAC AT GTGCACTCACf: AGTCTCACAC AT/delAT GTGCACTCAC 18) (SNP 18) (SEQ ID No: 227)(SEQ ID No: 229) r: GTGAGTGCAC AT GTGTGAGACT r: GTGAGTGCAC AT/delATGTGTGAGACT (SEQ ID No: 228) (SEQ ID No: 230) mut/mut: f: AGTCTCACACdelAT GTGCACTCAC (SEQ ID No: 231) r: GTGAGTGCAC delAT GTGTGAGACT(SEQ ID No: 232) wt/mut: Exon 19 (intron 34215 (SNP f: CATGTGCACT GACGTGGCCGG f: CATGTGCACT G/C ACGTGGCCGG 18) 17) (SEQ ID No: 233)(SEQ ID No: 235) r: CCGGCCACGT C AGTGCACATG r: CCGGCCACGT C/G AGTGCACATG(SEQ ID No: 234) (SEQ ID No: 236) mut/mut: f: CATGTGCACT C ACGTGGCCGG(SEQ ID No: 237) r: CCGGCCACGT G AGTGCACATG (SEQ ID No: 238) wt/mut:Exon 22 (intron 55156 (SNP f: GGGGCTGGGG C TGGGTGCGTG f: GGGGCTGGGGC/insTGGGGC TGGGTGCGTG 21) 28) (SEQ ID No: 239) (SEQ ID No: 241)r: CACGCACCCA G CCCCAGCCCC r: CACGCACCCA G/insGCCCCA CCCCAGCCCC(SEQ ID No: 240) (SEQ ID No: 242) mut/mut: f: GGGGCTGGGGC insTGGGGCTGGGTGCGTG (SEQ ID No: 243) r: CACGCACCCA insGCCCCA GCCCCAGCCCC(SEQ ID No: 244) Accession number AC003026 wt/mut: Exon 22 (intron55472 (SNP f: TGTCTAATTA T AGAAATGGAT f: TGTCTAATTA T/C AGAAATGGAT 22)27) (SEQ ID No: 245) (SEQ ID No: 247) r: ATCCATTTCT A TAATTAGACAr: ATCCATTTCT A/G TAATTAGACA (SEQ ID No: 246) (SEQ ID No: 248) mut/mut:f: TGTCTAATTA C AGAAATGGAT (SEQ ID No: 249) r: ATCCATTTCT G TAATTAGACA(SEQ ID No: 250) Accession number U91318 wt/mut: Exon 28 14008 (SNPf: CTGGGAAGTC G TCCCTGACCC f: CTGGGAAGTC G/A TCCCTGACCC 23)(SEQ ID No: 251) (SEQ ID No: 253) r: GGGTCAGGGA C GACTTCCCAGr: GGGTCAGGGA C/T GACTTCCCAG (SEQ ID No: 252) (SEQ ID No: 254) mut/mut:f: CTGGGAAGTC A TCCCTGACCC (SEQ ID No: 255) r: GGGTCAGGGA T GACTTCCCAG(SEQ ID No: 256) Accession number AC025277 wt/mut: Exon 29 (intron150727 f: CCATGTCAGC G TGACACAGGT f: CCATGTCAGC G/A TGACACAGGT 28)(SNP 24) (SEQ ID No: 257) (SEQ ID No: 259) r: ACCTGTGTCA C GCTGACATGGr: ACCTGTGTCA C/T GCTGACATGG (SEQ ID No: 258) (SEQ ID No: 260) mut/mut:f: CCATGTCAGC A TGACACAGGT (SEQ ID No: 261) r: ACCTGTGTCA T GCTGACATGG(SEQ ID No: 262) Accession number U91318 wt/mut: Exon 30 (intron17970 (SNP f: CTGGTTTTTT T CTTCCGGTCA f: CTGGTTTTTT T/delT CTTCCGGTCA29) 15) (SEQ ID No: 263) (SEQ ID No: 265) r: TGACCGGAAG A AAAAAACCAGr: TGACCGGAAG A/delA AAAAAACCAG (SEQ ID No: 264) (SEQ ID No: 266)mut/mut: f: CTGGTTTTTT delT CTTCCGGTCA (SEQ ID No: 267) r: TGACCGGAAGdelA AAAAAACCAG (SEQ ID No: 268) Accession number U91318 wt/mut:Exon 30 (intron 18195 (SNP f: CACTGGCACA G TGGCCTCTAG f: CACTGGCACA G/ATGGCCTCTAG 30) 16) (SEQ ID No: 269) (SEQ ID No: 271) r: CTAGAGGCCA CTGTGCCAGTG r: CTAGAGGCCA C/T TGTGCCAGTG (SEQ ID No: 270)(SEQ ID No: 272) mut/mut: f: CACTGGCACA A TGGCCTCTAG (SEQ ID No: 273)r: CTAGAGGCCA T TGTGCCAGTG (SEQ ID No: 274) wt/mut: Exon 31 (3′21133 (SNP f: CCCAAAACAC G CACACCCTGC f: CCCAAAACAC G/A CACACCCTGCflanking region) 29) (SEQ ID No: 275) (SEQ ID No: 277) r: GCAGGGTGTG CGTGTTTTGGG r: GCAGGGTGTG C/T GTGTTTTGGG (SEQ ID No: 276)(SEQ ID No: 278) mut/mut: f: CCCAAAACAC A CACACCCTGC (SEQ ID No: 279)r: GCAGGGTGTG T GTGTTTTGGG (SEQ ID No: 280) Accession number AC003026wt/mut: Exon 19 (intron 34218 (SNP f: GTGCACTCAC G TGGCCGGGTGf: GTGCACTCAC G/A TGGCCGGGTG 18) 38) (SEQ ID No: 281) (SEQ ID No: 283)(only in r: CACCCGGCCA C GTGAGTGCAC r: CACCCGGCCA C/T GTGAGTGCAC RCC(SEQ ID No: 282) (SEQ ID No: 284) samples) mut/mut: f: GTGCACTCAC ATGGCCGGGTG (SEQ ID No: 285) r: CACCCGGCCA T GTGAGTGCAC (SEQ ID No: 286)Accession number U91318 wt/mut: Exon 30 18067 (SNP f: CCACGGCAGC CGTGGACCTGG f: CCACGGCAGC C/T GTGGACCTGG 39) (SEQ ID No: 287)(SEQ ID No: 289) (only in r: CCAGGTCCAC G GCTGCCGTGG r: CCAGGTCCAC G/AGCTGCCGTGG RCC (SEQ ID No: 288) (SEQ ID No: 290) samples) mut/mut:f: CCACGGCAGC T GTGGACCTGG (SEQ ID No: 291) r: CCAGGTCCAC A GCTGCCGTGG(SEQ ID No: 292) Accession number U07050 wt/mut: Promoter 440 (SNPf: CTCCTTCCCT C GCTAGGTCCT f: CTCCTTCCCT C/T GCTAGGTCCT fragment 5 40)(SEQ ID No: 293) (SEQ ID No: 295) (only in r: AGGACCTAGC G AGGGAAGGAGr: AGGACCTAGC G/A AGGGAAGGAG RCC (SEQ ID No: 294) (SEQ ID No: 296)samples) mut/mut: f: CTCCTTCCCT T GCTAGGTCCT (SEQ ID No: 297)r: AGGACCTAGC A AGGGAAGGAG (SEQ ID No: 298) wt/mut: Promoter 1625 (SNPf: GGGAATCACT C AACCTCTCTG f: GGGAATCACT C/A AACCTCTCTG fragment 2 41)(SEQ ID No: 299) (SEQ ID No: 301) (only in r: CAGAGAGGTT G AGTGATTCCCr: CAGAGAGGTT G/T AGTGATTCCC RCC (SEQ ID No: 300) (SEQ ID No: 302)samples) mut/mut: f: GGGAATCACT A AACCTCTCTG (SEQ ID No: 303)r: CAGAGAGGTT T AGTGATTCCC (SEQ ID No: 304) Accession number U91318wt/mut: Exon 30 (intron 17900 (SNP f: TGTCTCCTTT C GCTTCTCCCAf: TGTCTCCTTT C/T GCTTCTCCCA 29) 42) (SEQ ID No: 305) (SEQ ID No: 307)(only in r: TGGGAGAAGC G AAAGGAGACA r: TGGGAGAAGC G/A AAAGGAGACA RCC(SEQ ID No: 306) (SEQ ID No: 308) samples) mut/mut: f: TGTCTCCTTT TGCTTCTCCCA (SEQ ID No: 309) r: TGGGAGAAGC A AAAGGAGACA (SEQ ID No: 310)Accession number AC026452 wt/mut: Promoter 38646 (SNP f: CCTTAAACAG GATTTGAAAAG f: CCTTAAACAG G/C ATTTGAAAAG fragment 1 32) (SEQ ID No: 311)(SEQ ID No: 313) r: CTTTTCAAAT C CTGTTTAAGG r: CTTTTCAAAT C/G CTGTTTAAGG(SEQ ID No: 312) (SEQ ID No: 314) mut/mut: f: CCTTAAACAG C ATTTGAAAAG(SEQ ID No: 315) r: CTTTTCAAAT G CTGTTTAAGG (SEQ ID No: 316) Accessionnumber AC025277 wt/mut: Exon 5 (intron 5) 33551 (SNP f: TGTGACCACA GATGAGTGTGT f: TGTGACCACA G/A ATGAGTGTGT 36) (SEQ ID No: 317)(SEQ ID No: 319) r: ACACACTCAT C TGTGGTCACA r: ACACACTCAT C/T TGTGGTCACA(SEQ ID No: 318) (SEQ ID No: 320) mut/mut: f: TGTGACCACA A ATGAGTGTGT(SEQ ID No: 321) r: ACACACTCAT T TGTGGTCACA (SEQ ID No: 322)

TABLE 3 New SNP's in the gene for MRP1 GI Seq Seq Seq Seq Site SNP Var.Pos. Acc no ID Forward¹ ID Reverse¹ ID IUB_Forward ID IUB_Reverse P3mrys546 a > g 51798 3582311 329 TAACCAGGTTgT 330 GAGGATCAAcA 331TAACCAGGTTr 332 GAGGATCAAyA TGATCCTC ACCTGGTTA TTGATCCTC ACCTGGTTA P1mryp282 g > a 37971 7363401 333 TGGGGTGGGGa 334 CCCCGCGCCAt 335TGGGGTGGGGr 336 CCCCGCGCCAy TGGCGCGGGG CCCCACCCCA TGGCGCGGGG CCCCACCCCAP1 mryp877 g > a 50892 3582311 337 TGGGCACGCGa 338 TGCGTGGGGG 339TGGGCACGCGr 340 TGCGTGGGGG CCCCCCACGCA GtCGCGTGCCC CCCCCCACGCAGyCGCGTGCCC A A E22 mryo336 g > a 55296 2815549 341 CCATGTGTCCa 342GAAGCCAGCGt 343 CCATGTGTCCr 344 GAAGCCAGCGy CGCTGGCTTC GGACACATGGCGCTGGCTTC GGACACATGG I21 mryo172 g > a 55132 2815549 345 TGAAGCCCCCa346 CCCACAAGGTt 347 TGAAGCCCCCr 348 CCCACAAGGTy ACCTTGTGGG GGGGGCTTCAACCTTGTGGG GGGGGCTTCA I21 mryo154 a > g 55114 2815549 349 TGGGTGGCACg350 TCACCAGCACc 351 TGGGTGGCACr 352 TCACCAGCACy GTGCTGGTGA GTGCCACCCAGTGCTGGTGA GTGCCACCCA I21 mryo152 a > g 55112 2815549 353 GCTGGGTGGCg354 ACCAAGCACTG 355 GCTGGGTGGCr 356 ACCAAGCACTG CAGTGCTGGT cGCCACCCAGCCAGTGCTGGT yGCCACCCAGC P1 mryp522 delCCCG 109 to 4826837 357 GGCCCGATCAC358 CGGCGGCGGG 359 GGCCCGATCAn 360 CGGCGGCGGG CCGCCC 122 CCGCCGCCGTGATCGGGCC CCCGCCGCCG nTGATCGGGCC GGTG P1 mryp491 delGCC 76 to 784826837 361 TCCCTGC 362 CGCTAGCGCT 363 TCCCTGC 364 CGCTAGCGCTn [GCC] ₁₃[GGC] ₁₃ [GCC] ₁₃ [GGC] ₁₃ AGCGCTAGCG GCAGGG nAGCGCTAGCG GCAGGG P1mryp489 del[GCC]₂ 73 to 78 4826837 365 TCCCTGC 366 CGCTAGCGCT 367TCCCTGC 368 CGCTAGCGCTn [GCC] ₁₂ [GGC] ₁₂ [GCC] ₁₂ [GGC] ₁₂ AGCGCTAGCGGCAGGGA nAGCGCTAGCG GCAGGGA P1 mryp486 del[GCC]₃ 70 to 78 4826837 369TCCCTGC 370 CGCTAGCGCT 371 TCCCTGC 372 CGCTAGCGCTn [GCC] ₁₁ [GGC] ₁₁[GCC] ₁₁ [GGC] ₁₁ AGCGCTAGCG GCAGGGA nAGCGCTAGCG GCAGGGA P1 mryp483del[GCC]₄ 67 to 78 4826837 373 TCCCTGC 374 CGCTAGCGCT 375 TCCCTGC 376CGCTAGCGCTn [GCC] ₁₀ [GGC] ₁₀ [GCC] ₁₀ GGC] ₁₀ AGCGCTAGCG GCAGGGAnAGCGCTAGCG GCAGGGA P1 mryp474 del[GCC]₇ 58 to 78 4826837 377 TCCCTGC378 CGCTAGCGCT 379 TCCCTGC 380 CGCTAGCGCTn [GCC] ₇ [GGC] ₇ [GCC] ₇ [GGC]₇ AGCGCTAGCG GCAGGGA nAGCGCTAGCG GCAGGGA I14 mrzl154 delAA 20097 to2815549 381 TCAAGCAGAGA 382 AACACTCTCTC 383 TCAAGCAGAGn 384 AACACTCTCTnC20098 GAGAGTGTT TCTGCTTGA AGAGAGTGTT TCTGCTTGA E9 mrzr176 c > t 603577209451 385 CTGGGGCCTTt 386 TGAATGACACa 387 CTGGGGCCTTy 388 TGAATGACACrGTGTCATTCA AAGGCCCCAG GTGTCATTCA AAGGCCCCAG I7 mrzs129 g > a 617867209451 389 ACACAAGGAGa 390 AACGGCTTCAt 391 ACACAAGGAGr 392 AACGGCTTCAyTGAAGCCGTT CTTCCTTGTGT TGAAGCCGTT CTCCTTGTGT I6 mrzu272 insC 76437/7209451 393 CAGGCCCCCCc 394 CCTGAGGTCTg 395 CAGGCCCCCCn 396 CCTGAGGTCTn76438 AGACCTCAGG GGGGGGCCTG AGACCTCAGG GGGGGGCCTG E2 mrzy349 g > a 395417209451 397 TACAGTTTTGaT 398 CTCAACAAAAt 399 TACAGTTTTGr 400 CTCAACAAAAyTTTGTTGAG CAAAACTGTA TTTTGTTGAG CAAAACTGTA ¹Brackets depict repeats.Numbers indicate how often the sequence in brackets is repeated.

TABLE 4 AA Seq Seq exchange ProtAccNo ID Protein mut ID Protein T73IGI:2828206 401 TPLNKiKTALG 402 TPLNKxKTALG A989T GI:2828206 403CNHVStLASNY 404 CNHVSxLASNY

1. A polynucleotide comprising a polynucleotide selected from the groupconsisting of: (a) a polynucleotide having the nucleic acid sequence ofSEQ ID NO: 75, 76, 81, 82, 87, 88, 93, 94, 99, 100, 105, 106, 111, 112,117, 118, 123, 124, 129, 130, 135, 136, 141, 142, 147, 148, 153, 154,159, 160, 165, 166, 171, 172, 177, 178, 183, 184, 189, 190, 195, 196,201, 202, 207, 208, 213, 214, 219, 220, 225, 226, 231, 232, 237, 238,243, 244, 249, 250, 255, 256, 261, 262, 267, 268, 273, 274, 279, 280,285, 286, 291, 292, 297, 298, 303, 304, 309, 310, 315, 316, 321, 322,329, 330, 333, 334, 337, 338, 341, 342, 345, 346, 349, 350, 353, 354,357, 358, 361, 362, 365, 366, 369, 370, 373, 374, 377, 378, 381, 382,385, 386, 389, 390, 393, 394, 397 or 398; (b) a polynucleotide encodinga polypeptide having the amino acid sequence of SEQ ID NO: 324, 326,328, 401 or 403; (c) a polynucleotide capable of hybridizing to a MRP-1gene, wherein said polynucleotide is having a substitution or deletionof at least one nucleotide at a position corresponding to position124667 of the MRP-1 gene (Accession No: AC026452), 1884, 1720 to 1723,1163, 926, 437, 381, 233, 189, 440 or 1625 of the MRP-1 gene (AccessionNo: U07050), 39508 of the MRP-1 gene (GI No: 7209451), 79, 88 or 249 ofthe MRP-1 gene (Accession No: AF022830), 95 or 259 of the MRP-1 gene(Accession No: AF022831), 57998, 57853 or 53282 of the MRP-1 gene (GINo: 7209451), 1377.10, 137667, 38646 or 137647 of the MRP-1 gene(Accession No: AC026452), 27159, 27258, 34206 to 34207, 34218, 34215,55156 or 55472 of the MRP-1 gene (Accession No: AC003026), 14008, 17970,18195, 21133, 18067, 17900 of the MRP-1 gene (Accession No: U91318), or150727 or 33551 of the MRP-1 gene (Accession No: AC025277), 174 of theMRP-1 gene (Accession No: AF022828), 248 or 258 of the MRP-1 gene(Accession No: AF022829), 51798 or 50892 of the MRP-1 gene (AccessionNo: GI 3582311), 37971 of the MRP-1 gene (Accession No: GI 7363401),55296, 55132, 55114, 55112 or 20097 to 20099 of the MRP-1 gene(Accession No: GI 2815549), 109 to 122, 76 to 78, 73 to 78, 70 to 78, 67to 78 or 58 to 78 of the MRP-1 gene (Accession No: GI 4826837), 60357,61786 or 39541 of the MRP-1 gene (Accession No: GI 7209451) or ainsertion of at least one nucleotide at a position corresponding toposition 55156/55157 of the MRP-1 gene (Accession No: AC003026) or437/438 or 926/927 of the MRP-1 gene (Accession No: U07050) or76437/76438 of the MRP-1 gene (Accession No: GI 7209451); (d) apolynucleotide capable of hybridizing to a MRP-1 gene, wherein saidpolynucleotide is having at a position corresponding to position 124667of the MRP-1 gene (Accession No: AC026452) a C, at a positioncorresponding to position 1884 of the MRP-1 gene (Accession No: U07050)a A, at a position corresponding to position 1720 to 1723 of the MRP-1gene (Accession No: U07050) a deletion, at a position corresponding toposition 1163 of the MRP-1 gene (Accession No: U07050) a T, at aposition corresponding to position 926/927 of the MRP-1 gene (AccessionNo: U07050) a insertion, at a position corresponding to position 437/438of the MRP-1 gene (Accession No: U07050) a insertion, at a positioncorresponding to position 381 of the MRP-1 gene (Accession No: U07050) aG, at a position corresponding to position 233 of the MRP-1 gene(Accession No: U07050) an A, at a position corresponding to position 189of the MRP-1 gene (Accession No: U07050) an A, at a positioncorresponding to position 39508 of the MRP-1 gene (GI No: 7209451) an A,at a position corresponding to position 174 of the MRP-1 gene (AccessionNo: AF022828) a T, at a position corresponding to position 248 of theMRP-1 gene (Accession No: AF022829) an A, at a position corresponding toposition 258 of the MRP-1 gene (Accession No: AF022829) a G, at aposition corresponding to position 79 of the MRP-1 gene (Accession No:AF022830) an A, at a position corresponding to position 88 of the MRP-1gene (Accession No: AF022830) a C, at a position corresponding toposition 249 of the MRP-1 gene (Accession No: AF022830) a G, at aposition corresponding to position 95 of the MRP-1 gene (Accession No:AF022831) a C, at a position corresponding to position 259 of the MRP-1gene (Accession No: AF022831) a G, at a position corresponding toposition 57998 of the MRP-1 gene (GI No: 7209451) a T, at a positioncorresponding to position 57853 of the MRP-1 gene (GI No: 7209451) a T,at a position corresponding to position 53282 of the MRP-1 gene (GI No:7209451) a G, at a position corresponding to position 137710 of theMRP-1 gene (Accession No: AC026452) a G, at a position corresponding toposition 137667 of the MRP-1 gene (Accession No: AC026452) a T, at aposition corresponding to position 137647 of the MRP-1 gene (AccessionNo: AC026452) a T, at a position corresponding to position 27159 of theMRP-1 gene (Accession No: AC003026) a C, at a position corresponding toposition 27258 of the MRP-1 gene (Accession No: AC003026) an A, at aposition corresponding to position 34206 to 34207 of the MRP-1 gene(Accession No: AC003026) a deletion, at a position corresponding toposition 34215 of the MRP-1 gene (Accession No: AC003026) a C, at aposition corresponding to position 55156/55157 of the MRP-1 gene(Accession No: AC003026) a insertion, at a position corresponding toposition 55472 of the MRP-1 gene (Accession No: AC003026) a C, at aposition corresponding to position 14008 of the MRP-1 gene (AccessionNo: U91318) an A, at a position corresponding to position 150727 of theMRP-1 gene (Accession No: AC025277) an A, at a position corresponding toposition 17970 of the MRP-1 gene (Accession No: U91318) a deletion, at aposition corresponding to position 18195 of the MRP-1 gene (AccessionNo: U91318) an A, at a position corresponding to position 21133 of theMRP-1 gene (Accession No: U91318) an A, at a position corresponding toposition 34218 of the MRP-1 gene (Accession No: AC003026) an A, at aposition corresponding to position 18067 of the MRP-1 gene (AccessionNo: U91318) a T, at a position corresponding to position 440 of theMRP-1 gene (Accession No: U07050) a T, at a position corresponding toposition 1625 of the MRP-1 gene (Accession No: U07050) an A, at aposition corresponding to position 17900 of the MRP-1 gene (AccessionNo: U91318) a T, at a position corresponding to position 38646 of theMRP-1 gene (Accession No: AC026452) a C, at a position corresponding toposition 33551 of the MRP-1 gene (Accession No: AC025277) an A, at aposition corresponding to position 51798 of the MRP-1 gene (AccessionNo: 3582311) an G, at a position corresponding to position 37971 of theMRP-1 gene (Accession No: 7363401) an A, at a position corresponding toposition 50892 of the MRP-1 gene (Accession No: 358231.1) an A, at aposition corresponding to position 55296 of the MRP-1 gene (AccessionNo: 2815549) an A, at a position corresponding to position 55132 of theMRP-1 gene (Accession No: 2815549) an A, at a position corresponding toposition 55114 of the MRP-1 gene (Accession No: 2815549) an G, at aposition corresponding to position 55112 of the MRP-1 gene (AccessionNo: 2815549) an G, at a position corresponding to position 109 to 122 ofthe MRP-1 gene (Accession No: 4826837) deletions, at a positioncorresponding to position 76 to 78 of the MRP-1 gene (Accession No:4826837) deletions, at a position corresponding to position 73 to 78 ofthe MRP-1 gene (Accession No: 4826837) deletions, at a positioncorresponding to position 70 to 78 of the MRP-1 gene (Accession No:4826837) deletions, at a position corresponding to position 67 to 78 ofthe MRP-1 gene (Accession No: 4826837) deletions, at a positioncorresponding to position 58 to 78 of the MRP-1 gene (Accession No:4826837) deletions, at a position corresponding to position 20097 to20099 of the MRP-1 gene (Accession No: 2815549) deletions, at a positioncorresponding to position 60357 of the MRP-1 gene (Accession No:7209451) a T, at a position corresponding to position 61786 of the MRP-1gene (Accession No: 7209451) an A, at a position corresponding toposition 76437/76438 of the MRP-1 gene (Accession No: 7209451) aninsertion or at a position corresponding to position 39541 of the MRP-1gene (Accession No: 7209451) an A; (e) a polynucleotide encoding anMRP-1 polypeptide or fragment thereof, wherein said polypeptidecomprises an amino acid substitution at position 329, 433 or 723 of theMRP-1 polypeptide (Accession No: P33527) or 73 or 989 of the MRP-1polypeptide (Accession No: GI 2828206); and (f) a polynucleotideencoding an MRP-1 polypeptide or fragment thereof, wherein saidpolypeptide comprises an amino acid substitution of Phe to Cys atposition 329, Arg to Ser at position 433 or Arg to Gln at position 723of the MRP-1 polypeptide (Accession No: P33527) or Thr to 11e atposition 73 or Ala to Thr at position 989 of the MRP-1 polypeptide(Accession No: GI 2828206).
 2. A polynucleotide of claim 1, wherein saidpolynucleotide is associated with a disease selected from the groupconsisting of cancer diseases and multidrug resistance related diseases.3. (canceled)
 4. A gene comprising the polynucleotide of any one ofclaim 1 or
 2. 5. (canceled)
 6. A vector comprising a polynucleotide ofany one of claims 1 to 2 or the gene of claim
 4. 7. (canceled)
 8. A hostcell genetically engineered with the polynucleotide of any one of claims1 to 2, the gene of claim 4 or the vector of claim
 6. 9. A method forproducing a molecular variant MRP-1 polypeptide or fragment thereofcomprising (a) culturing the host cell of claim 8; and (b) recoveringsaid protein or fragment from the culture.
 10. A method for producingcells capable of expressing a molecular variant MRP-1 polypeptidecomprising genetically engineering cells with the polynucleotide of anyone of claims 1 to 2, the gene of claim 4 or the vector of claim
 6. 11.A polypeptide or fragment thereof encoded by the polynucleotide of anyone of claims 1 to 2, the gene of claim 4 or obtainable by the method ofclaim 9 or from cells produced by the method of claim
 10. 12. Anantibody which binds specifically to the polypeptide of claim
 11. 13.The antibody of claim 12 which specifically recognizes an epitopecontaining one or more amino acid substitution(s) resulting from anucleotide exchange as defined in claim
 1. 14-16. (canceled)
 17. A solidsupport comprising one or a plurality of the polynucleotide of any oneof claims 1 to 2, the gene of claim 4 or the vector of claim 6, thepolypeptide of claim 11, the antibody of claim 12 or 13 or the host cellof claim 8 in immobilized form.
 18. (canceled)
 19. An in vitro methodfor identifying a single nucleotide polymorphism said method comprisingthe steps of: (a) isolating a polynucleotide of anyone claims 1 to 2 orthe gene of claim 4 from a plurality of subgroups of individuals,wherein one subgroup has no prevalence for a MRP-1 associated diseaseand at least one or more further subgroup(s) do have prevalence for aMRP-1 associated disease; and (b) identifying a single nucleotidepolymorphism by comparing the nucleic acid sequence of saidpolynucleotide or said gene of said one subgroup having no prevalencefor a MRP-1 associated disease with said at least one or more furthersubgroup(s) having a prevalence for a MRP-1 associated disease.
 20. Amethod for identifying and obtaining a pro-drug or a drug capable ofmodulating the activity of a molecular variant of a MRP-1 polypeptidecomprising the steps of: (a) contacting the polypeptide of claim 11, thesolid support of claim 17, a cell expressing a molecular variant genecomprising a polynucleotide of any one of claims 1 to 2, the gene ofclaim 4 or the vector of claim 6 in the presence of components capableof providing a detectable signal in response to drug activity with acompound to be screened for pro-drug or drug activity; and (b) detectingthe presence or absence of a signal or increase or decrease of a signalgenerated from the pro-drug or the drug activity, wherein the absence,presence, increase or decrease of the signal is indicative for aputative pro-drug or drug. 21-28. (canceled)
 29. A method of diagnosinga disorder related to the presence of a molecular variant of a MRP-1gene or susceptibility to such a disorder comprising determining thepresence of a polynucleotide of any one of claims 1 to 2 or the gene ofclaim 4 in a sample from a subject.
 30. (canceled)
 31. A method ofdiagnosing a disorder related to the presence of a molecular variant ofa MRP-1 gene or susceptibility to such a disorder comprising determiningthe presence of a polypeptide of claim 11 or the antibody of any one ofclaims 12 to 13 in a sample from a subject.
 32. The method of claim 29or claim 31, wherein said disorder is a cancer disease or a diseaserelated to multidrug resistance.
 33. (canceled)
 34. A method ofdetection of the polynucleotide of any one of claims 1 to 2 or the geneof claim 4 in a sample comprising the steps of (a) contacting a solidsupport of claim 17 or 18 with the sample under conditions allowinginteraction of the polynucleotide of claims 1 to 2 or the gene of claim4 with the immobilized targets on a solid support and; (b) determiningthe binding of said polynucleotide or said gene to said immobilizedtargets on a solid support.
 35. An in vitro method for diagnosing adisease comprising the steps of the method of claim 34, wherein bindingof said polynucleotide or gene to said immobilized targets on said solidsupport is indicative for the presence or the absence of said disease ora prevalence for said disease.
 36. A diagnostic composition comprisingthe polynucleotide of any one of claims 1 to 2, the gene of claim 4, thevector of claim 6, the polypeptide of claim 11 or the antibody of anyone of the claims 12 to
 13. 37-41. (canceled)
 42. A diagnostic kit fordetection of a single nucleotide polymorphism comprising thepolynucleotide of any one of claims 1 to 2, the gene of claim 4, thevector of claim 6, the polypeptide of claim 11, the antibody of any ofthe claims 12 to 13, the host cell of claim 8, or the solid support ofclaim 17.